Test strip with flared sample receiving chamber

ABSTRACT

A test strip with a sample receiving chamber having a novel flared portion that terminates in a sample receiving opening. The flared portion provides a reservoir from which sample fluid can be drawn into the capillary or sample receiving chamber. The wider opening provided by the present invention is easier to “target” with a sample fluid. In preferred embodiments, the hydrophilic reagent layer extends to the dosing end or side of the test strip and further promotes wicking of the sample into the sample receiving chamber and thus reduces dose hesitation. In other preferred embodiments, a tapered dosing end is provided on the test strip in combination with the flared portion, and this combination create a test strip that will draw sample fluid into the sample receiving chamber regardless of where along the dosing edge of the test strip the fluid sample makes contact.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/480,397, filed Jun. 20, 2003.

FIELD OF THE INVENTION

The present invention relates generally to the testing of body fluidsfor concentration of analytes and more particularly to a test strip orbiosensor for such testing.

BACKGROUND

Test strips are often used to measure the presence and/or concentrationsof selected analytes in test samples. For example, a variety of teststrips are used to measure glucose concentrations in blood to monitorthe blood sugar level of people with diabetes. These test strips includea reaction chamber into which a reagent composition has been deposited.Current trends in test strips require smaller test samples and fasteranalysis times. This provides a significant benefit to the patient,allowing the use of smaller blood samples that can be obtained from lesssensitive areas of the body. Additionally, faster test times and moreaccurate results enable patients to better control their blood sugarlevel.

In connection with smaller sample volumes, it is known to provide teststrips having a sufficiently small reaction chamber such that samplefluid is drawn therein by capillary action, which is a phenomenonresulting from the surface tension of the sample fluid and thethermodynamic tendency of a liquid to minimize its surface area. Forexample, U.S. Pat. No. 5,141,868 discloses a test strip having a cavitysized sufficiently small to draw sample liquid therein by capillaryaction. The cavity is defined by two parallel plates spaced about 1 mmapart by two epoxy strips extending lengthwise along lateral sides ofthe plates. The cavity is open at both ends, one of which receives thesample, and the other of which allows air to escape. The cavity includesan electrode structure and carries a coating of a material appropriateto the test to be performed by the test strip.

Various other test strip designs include capillary cavities that drawsample fluid therein and include vent openings to allow air to escape.As one should appreciate, capillary channels in current test stripdesigns are typically very small and are continually being designedsmaller to reduce the amount of sample needed for testing. However, thesmaller the capillary entrance width, the more difficult it becomes toaccurately apply (or “target”) a small sample volume to the capillary ofthe test strip. Targeting is even more important in segments of thedemographic with impaired vision and/or reduced dexterity because it ismore difficult for this segment to accurately align their fingers withthe dosing edge of a test strip. Furthermore, the sample fluid sometimesundesirably hesitates before being drawn into the capillary, aphenomenon referred to as “dose hesitation.” It would be desirable toovercome the difficulties associated with small capillaries in teststrip design.

SUMMARY OF THE INVENTION

The present invention provides a test strip with a sample receivingchamber having a novel flared portion that terminates in a samplereceiving opening. The flared portion provides a reservoir from whichsample fluid can be drawn into the capillary or sample receivingchamber, aids the user in introducing the sample to the test device, andreduces dose hesitation. In preferred embodiments, the hydrophilicreagent layer extends to the dosing end or side of the test strip andfurther promotes wicking of the sample into the sample receiving chamberand thus further reduces dose hesitation.

In one form thereof, the present invention provides a test stripcomprising a base substrate and a covering layer overlying the basesubstrate, the covering layer further comprising a vent. A spacing layeris disposed between the covering layer and the base substrate, and thespacing layer has a void that defines a sample receiving chamberdisposed between the base substrate and the covering layer. The samplereceiving chamber defines a flared portion that terminates in a fluidreceiving opening. The vent is in communication with the samplereceiving chamber, whereby air can escape from the vent as fluid isdrawn into the sample receiving chamber.

In a preferred form thereof, the covering layer, the base substrate andthe spacing layer are substantially flat, such that the sample receivingchamber comprises a substantially constant height and a width thatvaries at the flared portion. More preferably, the sample receivingchamber includes an elongated portion having a substantially constantwidth extending inwardly from the flared portion. The covering layer andthe base substrate include substantially aligned edges that comprise afluid receiving end or side of the test strip at which the fluidreceiving opening is located.

In another preferred form, the aligned edges comprise a notch whichfurther defines the opening. The notch is smaller than the flaredportion and is disposed centrally with respect to the flared portion.

In another preferred form, at least one electrode and a reagent layerare disposed in the sample receiving chamber, and the reagent layercovers at least one electrode. Most preferably, the reagent layerextends to the sample receiving opening.

In another preferred form, the present invention provides a test stripcomprising a base substrate having a reagent layer disposed thereon. Acovering layer overlies the base substrate and a sample receivingchamber is disposed between the base substrate and the covering layer.The sample receiving chamber comprises a flared portion defining asample receiving opening, and the reagent layer extends to the samplereceiving opening.

In a preferred form thereof, the test strip includes a slot incommunication with the sample receiving chamber, the slot defining avent opening in the covering layer that allows air to escape as fluidenters the sample receiving chamber. The sample receiving chambercomprises an elongated portion extending inwardly from the flaredportion. The flared portion is defined by a pair of opening walls thatnarrow in a direction toward the elongated portion, whereas theelongated portion is defined by substantially parallel walls that areconnected by an end wall.

In another form thereof, the present invention provides a method ofmaking a test strip. A base substrate, a spacing layer and a coveringlayer are provided. A void is formed in the spacing layer, the voidhaving an elongated portion and a bulbous portion. The spacing layer islaminated to the base substrate and the covering layer is laminated tothe spacing layer, thereby forming a test strip precursor. A cut is madethrough the precursor to make the test strip, the cut crossing thebulbous portion of the void and forming a sample receiving edge of thetest strip, wherein the void defines a sample receiving chamber having aflared portion terminating in a sample receiving opening at the samplereceiving edge of the test strip.

In a preferred form of this inventive method, the sample receiving edgecomprises an end of the test strip, the sample receiving opening isflared outwardly, and the dosing end of the test strip is tapered.

The present invention provides a very easy-to-dose test strip andprovides a robust but flexible manufacturing process. The various otherfeatures that characterize the invention are pointed out withparticularity in the attached claims. For a better understanding of theinvention, its advantages, and objectives obtained therefrom, referenceshould be made to the drawings and to the accompanying description, inwhich there is illustrated and described preferred embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the drawings, wherein like reference numerals andletters indicate corresponding structure throughout the several views:

FIG. 1 is a perspective view of a test strip or biosensor in accordancewith the present invention.

FIG. 1A is an enlarged fragmentary perspective view of the test stripshown in FIG. 1, illustrating one embodiment of the novel vent openingor slot.

FIG. 1B is an enlarged fragmentary perspective view illustrating analternate embodiment of the vent opening or slot in accordance with thepresent invention.

FIG. 1C is an enlarged fragmentary perspective view illustrating anotheralternate embodiment of the vent opening or slot and also illustratingan alternate configuration of the opening to the sample receivingchamber of the biosensor in accordance with the present invention.

FIG. 2 is an exploded, perspective view of the biosensor of FIG. 1.

FIG. 3 is a cross-sectional view of a portion of the biosensor of FIG.1, additionally illustrating adhesive layers that have been omitted fromFIGS. 1-2.

FIG. 4 is a top, plan view of a portion of the biosensor of FIG. 1, withportions broken away to show underlying details.

FIGS. 5 and 5A show a process flow diagram for a method for producing abiosensor in accordance with the present invention.

FIG. 6 is a perspective view showing the reel-to-reel processing andcutting of a web material useful in forming the bottom substrate of thebiosensor of the present invention.

FIG. 7 is a perspective view of a portion of a webbing, showing anexemplary pattern of electrical components on the base substrate.

FIG. 8 is a perspective view of a portion of the webbing of FIG. 7 andincluding a reagent composition coated thereon.

FIG. 9 is an exploded, perspective view showing a spacing layer and theassociated adhesive layers and release liners.

FIG. 10 is an exploded perspective view of a portion of the spacinglayer with pre-capillary chambers cut out and the spacing layer beingaligned for lamination to a base substrate having electrode patternsthereon.

FIG. 11 is a perspective view of an assembly of the base substrate withthe spacing layer.

FIG. 12 is an exploded, perspective view showing the combination of thebody and chamber covers for assembly onto the base substrate and spacinglayer.

FIG. 13 is a perspective view of a portion of an assembly including theseveral layers comprising the biosensor.

FIG. 14 is a perspective view of a portion of webbing including severaldetachable biosensors.

FIG. 15 is a perspective view of a single biosensor separated from theassembled webbing.

FIG. 16A is a top view of a single biosensor separated from assembledwebbing.

FIG. 16B is a fragmentary top view of a web or test strip precursorillustrating a void with a flared end that will ultimately form thesample receiving chamber for a biosensor such as that shown in FIG. 16A.

FIG. 16C is a fragmentary top view of a web or test strip precursorillustrating a light bulb shaped void that will ultimately form thesample receiving chamber for a biosensor such as that shown in FIG. 16A.

FIG. 17A is a top view of a single biosensor separated from assembledwebbing.

FIG. 17B is a fragmentary top view of a web or test strip precursorillustrating a chalice shaped void that will ultimately form the samplereceiving chamber for a biosensor such as that shown in FIG. 17A.

FIG. 17C is a fragmentary top view of a web or test strip precursorillustrating a keyhole shaped void that will ultimately form the samplereceiving chamber for a biosensor such as that shown in FIG. 17A.

FIG. 18A is a top view of a single biosensor separated from assembledwebbing.

FIG. 18B is a fragmentary top view of a web or test strip precursorillustrating a T-shaped void that will ultimately form the samplereceiving chamber for a biosensor such as that shown in FIG. 18A.

FIG. 18C is a fragmentary top view of a web or test strip precursorillustrating a T-shaped void that will ultimately form the samplereceiving chamber for a biosensor such as that shown in FIG. 18A.

FIG. 19A is a fragmentary top view of a single test strip illustrating aY-shaped flared portion leading inwardly from a straight dosing edge ofthe test strip to an elongated portion.

FIG. 19B is a fragmentary top view of a single test strip illustrating acurved profile for a dosing edge and a curved shaped flared portionleading inwardly on the test strip to an elongated portion.

FIG. 19C is a fragmentary top view of a single test strip illustrating acurved concave profile for a dosing edge and a Y-shaped flared portionleading inwardly on the test strip to an elongated portion.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the specific embodimentsillustrated herein and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the invention is thereby intended. Any alterations and furthermodifications in the described processes or devices, and any furtherapplications of the principles of the invention as described herein, arecontemplated as would normally occur to one skilled in the art to whichthe invention relates.

System

The present invention relates to a system that is useful for assessingan analyte in a sample fluid. The system includes devices and methodsfor evaluating the sample fluid for the target analyte. As more fullydescribed hereafter, the evaluation may range from detecting thepresence of the analyte to determining the concentration of the analyte.The analyte and the sample fluid may be any for which the test system isappropriate. For purposes of explanation only, a preferred embodiment isdescribed in which the analyte is glucose and the sample fluid is bloodor interstitial fluid. However, the present invention clearly is not solimited in scope.

Sensor

One component of the system is an electrochemical sensor including asample-receiving chamber for the sample fluid, and a suitable reagentfor producing an electrochemical signal in the presence of the testanalyte. The sensor preferably comprises a disposable test strip,particularly one having a laminar construction providing an edge openingwhich communicates with the sample-receiving chamber.

The reagent is disposed within the sample-receiving chamber in positionto provide the electrochemical signal to a working electrode alsopositioned within the chamber. In appropriate circumstances, such as forglucose detection, the reagent may contain an enzyme and optionally amediator.

Meter

The sensor is used in combination with a meter for determination of theanalyte in the sample fluid. The meter conventionally includes aconnection with the electrodes of the sensor and circuitry to evaluatethe electrochemical signal corresponding to the concentration of theanalyte. The meter may also include means for determining that thesample fluid has been received by the sensor, and that the amount ofsample fluid is sufficient for testing. The meter typically will storeand display the results of the analysis, or may alternatively providethe data to a separate device.

Analyte—Characteristic

The system can provide either a qualitative or quantitative indicationfor the analyte. In one embodiment, the system indicates simply thepresence of the analyte in the sample fluid. The system may also providea reading of the quantity or concentration of the analyte in the samplefluid. In a preferred embodiment, it is a feature of the presentinvention that a highly accurate and precise reading of the analyteconcentration is quickly obtained from a small volume of sample fluid.

Analyte—Type

The system is useful for the determination of a wide variety ofanalytes. The test strip, for example, is readily adapted for use withany suitable chemistry that can be used to assess the presence of theanalyte. Most preferably, the system is configured and used for thetesting of an analyte in a biological fluid. Such analytes may include,for example, glucose, cholesterol, HDL cholesterol, triglycerides,lactates, lactate dehydrogenase, alcohol, uric acid, and 3-hydroxybutricacid (ketone bodies). Commensurate modifications to the system will beapparent to those skilled in the art. For purposes of explanation, andin a particularly preferred embodiment, the system is described withrespect to the detection of glucose in a biological fluid.

Interferants

Test methodologies may be variously affected by the presence ofinterferants in the sample fluid. For example, the testing for glucosein a blood sample may be impacted by such factors as oxygen, bilirubin,hematocrit, uric acid, ascorbate, acetaminophen, galactose, maltose, andlipids. The present system is adaptable to minimize or eliminate theadverse effects of interferants that may also be present in the samplefluid. These effects may be addressed by appropriate selection of testmaterials and parameters, such as by the selection of chemistries thatare known to be impacted less, or not at all, by possible interferants.As is known in the art, other steps may also be taken to addresspossible interferant effects, such as the use of coatings or films thatprevent the interferant from entering the test zone. In addition,modifications to the electrode configurations or interrogation methodscan be used to minimize the effect of interferants.

Fluid Type

The system is useful with a wide variety of sample fluids, and ispreferably used for the detection of analytes in a biological fluid. Inthis context, the term “biological fluid” includes any bodily fluid inwhich the analyte can be measured, for example, interstitial fluid,dermal fluid, sweat, tears, urine, amniotic fluid, spinal fluid andblood. The term “blood” in the context of the invention includes wholeblood and its cell-free components, namely plasma and serum. Inaddition, the system is useful in connection with control fluids thatare used in conventional fashion to verify the integrity of the systemfor testing.

In a preferred embodiment, the system is employed for the testing ofglucose. The sample fluid in this instance may specifically include, forexample, fresh capillary blood obtained from the finger tip or approvedalternate sites (e.g., forearm, palm, ear lobe, upper arm, calf andthigh), fresh venous blood, and control solutions supplied with or forthe system.

The fluid may be acquired and delivered to the test strip in anyfashion. For example, a blood sample may be obtained in conventionalfashion by incising the skin, such as with a lancet, and then contactingthe test strip with fluid that appears at the skin surface. It is anaspect of the present invention that the test strip is useful with verysmall fluid samples. It is therefore a desirable feature of theinvention that only a slight incising of the skin is necessary toproduce the volume of fluid required for the test, and the pain andother concerns with such method can be minimized or eliminated.

It is also well known that different locations on the skin will producemore or less amounts of blood upon lancing. The finger tip, for example,is a commonly used site for obtaining a blood sample because it producesa relatively large amount of blood upon lancing. However, it is alsoknown that areas that produce larger volumes of blood are generallyassociated with greater degrees of pain for the user. It is therefore anadditional advantage of the present system that the required volume ofsample fluid is sufficiently small that the test strip is useful withthe amount of blood typically obtained upon lancing less productive, butalso less painful, areas of the skin, such as the palm and upper arm.The use of these locations to obtain sample fluids for testing issometimes referred to as “alternate site testing”. The present inventionis particularly well suited to use with sample fluids, e.g., blood orinterstitial fluid, obtained at these alternate sites.

Test Strip—General

Introduction.

The test strip includes several basic components. The strip comprises asmall body defining a chamber in which the sample fluid is received fortesting. This “sample-receiving chamber” is filled with the sample fluidby suitable means, preferably by capillary action, but also optionallyassisted by pressure or vacuum. The sample-receiving chamber includeselectrodes and chemistry suitable for producing an electrochemicalsignal indicative of the analyte in the sample fluid.

Basic Description.

Referring in particular to the drawings, there is shown a preferredembodiment of a test strip useful in accordance with the presentinvention. The test strip 10 includes a base substrate 12, a spacinglayer 14 and a covering layer 16 comprising body cover 18 and chambercover 20. The spacing layer 14 includes a void portion 22 to provide asample-receiving chamber 24 extending between the base substrate 12 andthe covering layer 16.

The base substrate 12 carries an electrode system 26 including aplurality of electrodes 28 and electrode traces 30 terminating incontact pads 32. The electrodes are defined as those portions ofelectrode traces 30 that are positioned within the sample-receivingchamber 24. Various configurations of the electrode system 26 may beused, as set forth hereafter. A suitable reagent system 33 overlies atleast a portion of the electrodes or electrode pairs 28 within thesample-receiving chamber.

The body cover 18 and the chamber cover 20 overlying the spacing layer16 define a slot 34 therebetween, the slot defining a vent openingcommunicating with the sample-receiving chamber to allow air to escapethe chamber as a sample fluid enters the chamber from the edge openingor fluid receiving opening 35. The test strip therefore includes adosing end 36 and a meter insertion end 38. The shape of the dosing endis typically distinguishable from the meter end so as to aid users. Asshown in FIG. 1, preferably, dosing end 36 is tapered or narrowed toform a trapezoidal shape to aid users in aligning the sample-receivingchamber 24 of test strip 10 with a fluid sample. The tapered shape ofthe dosing end 36 minimizes the available contact surface of the dosingend to the user's skin, thereby aiding the alignment of thesample-receiving chamber 24 with the fluid sample, as described in moredetail below.

In addition, strip graphics and contrasting colors at the dosing end arepreferably used to further improve the intuitiveness of the stripdesign. Similarly, at the meter insertion end, chevron 31 indicates thedirection of insertion of the strip into the meter. Further, chevron 31is sized and positioned on the test strip 10 such that the chevron 31 isinside the meter, and therefore hidden from view, when the test strip 10is properly inserted into the meter. The size and position of chevron 31(as opposed to an arrow) lessens the likelihood that users will jam atest strip marked with the chevron 31 into the meter and damage ordestroy the test strip 10.

General Dimensions.

The test strip is a relatively small device that is dimensioned forcompactness and ease of storage and use. In a typical embodiment, thestrip length is on the order of 20 to 50 mm, preferably about 33 toabout 38 mm, in length, and 5 to 15 mm, preferably about 7 to about 9mm, in width. The distance from the slot or vent opening 34 to the edgeof the meter is sized to provide a “grab area” where there is no bloodpresent, and to guard against blood contamination of the meter contactarea, and therefore may be in the range of 5 to 35 preferably ≧13 mm.The length of the test strip portion (from the meter insertion end 38)that is inserted into the meter is preferably ≦6.0 mm along the longaxis of the test strip.

The preferred laminar construction of the test strip also provides adevice that is relatively thin. The minimal thickness of the stripallows ready packaging of the strip in appropriate containers that areconvenient for the user. For example, the overall thickness of the teststrip may be about 500 to 525 μm. The.thickness of the test stripportion that is inserted into the meter contact may be about 250 μm.

Substrate

The test strip includes a base substrate 12 which comprises aninsulating material supporting the electrode system and othercomponents. Typically, plastics such as vinyl polymers, polyimides,polyesters, and styrenes provide the electrical and structuralproperties which are required. Further, because the test strip ispreferably mass producible from rolls of material, it is desirable thatthe material properties be appropriate to have sufficient flexibilityfor roll processing, while also giving a useful stiffness to thefinished strip. The base substrate can be selected as a flexiblepolymeric material such as polyester, especially high temperaturepolyester materials; polyethylene naphthalate (PEN); and polyimide, ormixtures of two or more of these. Polyimides are available commercially,for example under the trade name Kapton®, from E.I. duPont de Nemoursand Company of Wilmington, Del. (duPont). A particularly preferred basesubstrate material is MELINEX® 329 available from duPont.

Electrodes

Type.

The invention relates to an “electrochemical sensor”, which is a deviceconfigured to detect the presence of, and/or measure the concentrationof, an analyte by way of electrochemical oxidation and reductionreactions within the sensor. These reactions are transduced to anelectrical signal that can be correlated to an amount or concentrationof the analyte. The test strip therefore includes an electrode system 26comprising a set of measuring electrodes, e.g., at least a workingelectrode and a counter electrode, within the sample-receiving chamber.The sample-receiving chamber is configured such that sample fluidentering the chamber is placed in electrolytic contact with both theworking electrode and the counter electrode. This allows electricalcurrent to flow between the measuring electrodes to effect theelectrooxidation or electroreduction of the analyte.

In the context of the present invention, a “working electrode” is anelectrode at which analyte is electrooxidized or electroreduced with orwithout the agency of a redox mediator. The term “counter electrode”refers herein to an electrode that is paired with the working electrodeand through which passes an electrochemical current equal in magnitudeand opposite in sign to the current passed through the workingelectrode. The term “counter electrode” is meant to include counterelectrodes which also function as reference electrodes (i.e.,counter/reference electrodes).

Electrode Material.

The working and counter electrodes, and the remaining portions of theelectrode system, may be formed from a variety of materials, as known inthe art. The electrodes should have a relatively low electricalresistance and should be electrochemically inert over the operatingrange of the test strip. Suitable conductors for the working electrodeinclude gold, palladium, platinum, carbon, titanium, ruthenium dioxide,and indium tin oxide, and iridium, as well as others. The counterelectrode may be made of the same or different materials, e.g.,silver/silver chloride. In a preferred embodiment, the working andcounter electrodes are both gold electrodes.

Electrode Application.

The electrodes may be applied to the base substrate in any fashion thatyields electrodes of adequate conductivity and integrity. Exemplaryprocesses are well known in the art, and include, for example,sputtering, printing, etc. In a preferred embodiment, gold electrodesare provided by coating the base substrate and then removing selectedportions of the coating to yield the electrode system. A preferredremoval method is laser ablation, and more preferably broad field laserablation.

Laser ablative techniques typically include ablating a single metalliclayer or a multi-layer composition that includes an insulating materialand a conductive material, e.g., a metallic-laminate of a metal layercoated on or laminated to an insulating material (discussed below). Themetallic layer may contain pure metals, alloys, oxides, or othermaterials, which are metallic conductors. Examples of metals ormetallic-like conductors include: aluminum, carbon (such as graphite),cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium,mercury (as an amalgam), nickel, niobium, osmium, palladium, platinum,rhenium, rhodium, selenium, silicon (such as highly dopedpolycrystalline silicon), silver, tantalum, tin, titanium, tungsten,uranium, vanadium, zinc, zirconium, mixtures thereof, and alloys orsolid solutions of these materials. Preferably, the materials areselected to be essentially unreactive to biological systems; suchmaterials include: gold, platinum, palladium, iridium, silver, or alloysof these metals or indium tin oxide. The metallic layer may be anydesired thickness. In a preferred embodiment, the thickness is about 50nm.

Configuration.

The electrode system may have a variety of configurations suited to theoperation of the test strip and meter. For any embodiment, the workingand counter electrodes preferably are positioned and dimensioned tominimize the volume of sample fluid required to cover them. It is alsopreferable that the electrodes be configured to maintain a current fluxof sufficient magnitude as to be measurable using a relativelyinexpensive hand-held meter.

By way of further example, a preferred embodiment includes a counterelectrode which extends around both sides of the working electrode. Thecounter electrode therefore has two elements, one in front of theworking electrode and the other behind the working electrode, as thesample fluid enters the sample-receiving chamber. More specifically, thecounter electrode includes elements 40 and 42 which extend across thesample-receiving chamber. Each of these elements is about 250 μm wide.The working electrode element 44 has a width of about 250 μm, and isspaced from each of the two counter electrode elements by about 255 μm.It will be appreciated that this is only one of a number ofconfigurations for the measuring electrodes.

The traces 30 and the contact pads 32 may be provided in a variety offashions consistent with their intended function relative to the teststrip. These components of the electrode system are preferably composedof the same material as the electrodes, and are preferably applied tothe base substrate in the same manner and simultaneously with theapplication of the electrodes. In a preferred embodiment, the traces andcontact pads are gold, and are formed by laser ablation, particularly asdescribed in U.S. patent application, Ser. No. 10/601,144, filed Jun.20, 2003, entitled, Method of Making a Biosensor, the disclosure ofwhich is hereby incorporated by reference. However, alternate materialsand methods of application may be employed.

Chemistry

Reagent Composition.

The test strip includes a chemical reagent within the sample-receivingchamber for reacting with the test analyte to produce theelectrochemical signal that represents the presence of the analyte inthe sample fluid. The reagent layer can include a variety of activecomponents selected to determine the presence and/or concentration ofvarious analytes. The test chemistry is therefore selected in respect tothe analyte to be assessed. As is well known in the art, there arenumerous chemistries available for use with each of various analytes.For example, in one preferred embodiment, the test strip of the presentinvention can include one or more enzymes, co-enzymes, and co-factors,which can be selected to determine the presence of glucose in blood. Theselection of an appropriate chemistry is therefore well within the skillin the art, and further description herein is not required in order toenable one to make and use the test strips with various analytes.

Adjuvants.

In conventional fashion, the reagent chemistry may include a variety ofadjuvants to enhance the reagent properties or characteristics. Forexample, the chemistry may include materials to facilitate the placementof the reagent composition onto the test strip and to improve itsadherence to the strip, or for increasing the rate of hydration of thereagent composition by the sample fluid. Additionally, the reagent layercan include components selected to enhance the physical properties ofthe resulting dried reagent layer, and the uptake of a liquid testsample for analysis. Examples of adjuvant materials to be used with thereagent composition include thickeners, viscosity modulators, filmformers, stabilizers, buffers, detergents, gelling agents, fillers, filmopeners, coloring agents, and agents endowing thixotropy.

In a preferred embodiment of the test sample, the majority of thechamber is hollow before use. In the very small sample chamber of thetest strips according to the present invention, it is preferable thatthe reagent layer be thin and uniform. Since the sample-receivingchamber is very small, less than about 1 μl, the depth or verticalheight of the chamber is small. Consequently, the reagent layer shouldnot occupy the majority of the internal cavity of the chamber. Thereagent layer should be sufficiently thin to leave ample space for thetest sample in the chamber. Further, the liquid test sample will hydrateor dissolve the thin reagent layer more quickly. As discussed in theabove reaction scheme, the mediator and mediator redox products diffusethrough and within the reagent layer/gradient to the electrodes. Thereactive components and intermediates will have a short distance todiffuse through a thin reagent, therefore, diffusion to the electrodeswill occur in less time. Additionally, the capture efficiency ofmediator redox products at an electrode will be greater for a thin layerof enzyme than a thick layer.

Conversely, a thick reagent layer will take more time for the liquidtest sample to hydrate or dissolve, and a thick reagent layer willincrease the time that it takes for the mediator/mediator redox productsto approach the electrodes. This can delay the time to determine theanalyte concentration and introduce errors into the determination.

It is preferred that the reagent layer have a uniform thickness.Thickness inhomogeneity can lead to variability in determining theanalyte concentration. In a preferred embodiment, the reagent layer hasa uniform thickness throughout the entire sample receiving chamber. Inthis preferred embodiment, the reagent layer is not thicker around theperimeter of the sample receiving chamber adjacent the vertical sidewalls that define the chamber than in the central portion of thechamber. Consequently, the reagent layer does not exhibit a meniscusprofile.

The reagent composition is formulated as a viscous solution that can bedeposited in a thin, uniform layer on the base layer. The reagentcomposition includes thickeners and thixotropic agents to enhance thephysical properties of the reagent layer. The thickeners are selected toprovide a thick, liquid matrix having the remaining componentshomogeneously dispersed therein. The thickening and thixotropic agentsalso inhibit the liquid or semi-paste material from running or spreadingover the surface of the base layer after it has been deposited andbefore it dries. After the reagent composition is deposited, it quicklydries to a readily hydratable matrix.

The reagent composition is provided to dry rapidly either with airdrying or heat drying. After drying, the deposited reagent layerexhibits a thickness of between about 1 micron and about 20 microns.More preferably, the dried reagent layer exhibits a thickness of betweenabout 2 microns and about 6 microns.

The reagent composition can be deposited on the test strip surface usinga variety of coating methods including slot-die coating, curtaincoating, hot melt coating, rotary screen coating, doctor blade or airknife coating, Meyer bar coating, and reverse roll coating techniques.These techniques are known to those skilled in the art. Preferably, thereagent layer is deposited on the flexible web as a wet composition at athickness of between about 40 μm and about 100 μm. More preferably, thereagent composition is deposited as a wet composition at a thickness ofbetween about 60 μm and about 80 μm. The composition may be applied as auniformly thin layer of a reagent directly on top of the measuringelectrodes and along the length of a web of multiple test strips, as acontinuous narrow band. In preferred embodiments, the narrow band has awidth of between about 7 mm and 8 mm and a dry thickness of betweenabout 3 um and about 20 um. The composition may also be applied ontoother electrodes that may reside in the sample-receiving chamber,depending on the desired functionality of such extraneous electrodes.

Spacing Layer

Configuration.

The test strip includes a spacing layer 14 which overlies the basesubstrate and which in part defines the sample-receiving chamber. Inparticular, the spacing layer 14 includes a void portion 22substantially defining the height and the perimeter of thesample-receiving chamber 24. The void portion 22 is conveniently placedto have an edge opening whereby the sample fluid is contacted with theedge opening to enter into the sample-receiving chamber. The edgeopening preferably is located at the end of the test strip, although itwill be appreciated that placement on a side edge is also useful.

Materials.

The spacing layer 14 may be made of any material useful for fabricationwith the test strip. Because the spacing layer partially defines theheight of the sample-receiving chamber, the material should havesufficient strength at thicknesses appropriate to the desired height ofthe chamber. Another function of the spacing layer is to protect theelectrode traces that extend along the upper surface of base substrate12. The material should also be readily attached to the base substrateand the cover materials, either by heat-sensitive or pressure-sensitiveadhesives, or other means, such as heat or laser welding. Examples ofsuitable materials include a 100 μm PET, or PEN foil coated or combinedwith adhesives such as ARCare 90132 from Adhesives Research Inc.

Covering Layer

Configuration.

A covering layer 16 is received over and attached to the spacing layer14. One function of the covering layer is to form the top surface of thesample-receiving chamber. Another function is the provision of ahydrophilic surface to aid in acquisition of the test sample. Inaddition, the covering layer 16 preferably defines a vent opening 34that allows air to escape from the interior of the chamber as the samplefluid enters and moves into the sample-receiving chamber.

The covering layer can be formed as a unitary piece with slot 34′ formedas a recess on the underside thereof, as shown in FIG. 1B. For massproduction purposes, slot 34′ would be substantially straight as shownand extend across the entire width of the test strip, such that airwould vent from the sample receiving chamber 24 to the vent openingsformed on opposite lateral sides of the test strip. However, the slotcould comprise a groove or recess that only extends from the chamber 24to one side of the test strip, although such configuration is notpreferred for mass production purposes.

Another alternate embodiment is shown in FIG. 1C, in which chamber cover20 “overlaps” body cover 18. In this arrangement, a small end portion 37of cover layer 20 is bent upwardly and extends across the edge of bodycover 18. A slot 34″ is thereby formed having roughly a triangularshaped cross section as can be seen at the edges of the strip, at whichthere are triangular shaped openings that allow air to escape. In this“overlap” arrangement, the precise placement of the chamber cover 20with respect to body cover 18 along the lengthwise direction of thestrip is not critically important. That is, the amount of chambercovering material overlapping body cover 18 can vary without affectingthe dimensions or placement of the slot. This has advantages inmanufacturing, as will become apparent with reference to the discussionbelow.

Preferably, body cover 18 and chamber cover 20 comprise two separatemembers for ease in fabrication and in forming the vent opening. Bodycover 18 and chamber cover 20 are both disposed in substantially thesame horizontal plane. The chamber cover 20 substantially covers thevoid portion 22 of the spacing layer, and forms the top of thesample-receiving chamber. The chamber cover preferably includes ahydrophilic coating or treatment 21 on its underside, as described inmore detail below. The body cover and the chamber cover are positionedend to end in the lengthwise direction along the test strip and includeslot 34 therebetween as shown in FIG. 1A. The slot is located adjacentthe interior end of the void portion 22 of the spacing layer, and in thepreferred embodiment in FIG. 1A, forms a small gap that spaces chambercover 20 from body cover 18. The gap constitutes the vent opening 34 incommunication with the sample-receiving chamber. Slot 34 issubstantially straight and extends across the width of test strip 10.Slot 34 is oriented substantially perpendicular to the longitudinal orlengthwise axis of test strip 10. Sample fluid entering thesample-receiving chamber will expel air through the vent opening definedby slot 34. If the slot be formed as a gap, some or most of the airexpelled will exit from the top of the test strip.

The slot is located at a position relative to the sample-receivingchamber that is interior of the location of the electrode system 26.Sample fluid entering the sample-receiving chamber will progress as faras the vent opening, but no further. When viewed from the top, the slotprovides a visual indication of a “fill-line,” as described herein. Theplacement of the vent opening therefore assures that sufficient samplefluid can be received to fully cover the electrode system. At the sametime, the placement of the vent opening will inhibit continued wickingof the sample fluid beyond the region of the electrode system.

The formation of the slot and vent opening by the spacing of the bodycover and the chamber cover is further advantageous because it avoidsthe need to otherwise form an aperture in the covering layer or baselayer. In the prior art, it has been an approach to form the ventopening by punching a hole in either the top or bottom film forming thesample-receiving chamber, which presents fabrication issues because ofthe need to precisely locate the hole relative to the sample-receivingchamber. While this approach is also suitable for a test strip, thepreferred design described herein avoids the need to align the ventopening laterally relative to the test strip. Further, the presentdesign is well suited to mass production of the test strips by rollprocessing techniques, as described hereafter.

At the same time, the vent construction may be made in a manner toinhibit the wicking of sample fluid laterally along the slot beyond themiddle area that overlies the sample receiving chamber 24. For example,the body cover is preferably secured to the spacing layer by means of anadhesive 46, as shown in FIG. 3. The use of a hydrophobic adhesive willinhibit blood, interstitial fluid, and other aqueous liquids from movingalong the laterally-extending slot by capillary action. The entire bodycover, or portions adjacent to the vent opening, may also be hydrophobicto inhibit wicking. Materials and methods for providing hydrophobicproperties for a surface of a material are well known in the art. Thechamber cover may be secured to the spacing layer by the same ordifferent adhesive than adhesive 46, as explained below.

Adhesive 49 secures the spacing layer to the base substrate 12. Adhesive46, as well as adhesive 49 and the material for spacing layer 14, areall formed of substantially hydrophobic material in the illustratedembodiment. As such, the vertical walls of the capillary chamber formedin strip 10 are hydrophobic. By contrast, the floor of the chamber iscovered with a hydrophilic reagent and the underside of layer 20 iscoated with a hydrophilic coating 21 (FIG. 2). In other words, thehorizontal surfaces in the capillary are hydrophilic while the verticalsurfaces are hydrophobic. This has been found to promote good wicking ofthe sample into the capillary chamber, yet prevents unwanted migrationof the sample laterally from the chamber, e.g., between the spacer layerand the base substrate.

Materials.

The body cover and chamber cover may be made of any materials useful forfabrication with the test strip. The materials for the body cover andthe chamber cover may be the same or different. The materials should bereadily attached to the spacing layer, either by heat-sensitive orpressure-sensitive adhesives, or other means such as heat or laserwelding. Examples of suitable materials for both the chamber cover andbody cover include approximately 127 μm thick foil of PET. The chambercover preferably includes a hydrophilic layer 21 as disclosed in WO02/085185, ARFlow® 90191 from Adhesives Research Inc.

The covering layer 16 may also be used to facilitate viewing of thesample fluid as it enters the sample-receiving chamber. This isaccomplished by providing a contrast in color or shading between thechamber and the surrounding area. For example, in one approach theportion of the spacing layer 14 that surrounds void 22 is provided witha color that contrasts with the color of the bottom of thesample-receiving chamber, e.g., the color of the chemical reagent layerpositioned at the bottom of the chamber. This contrasting color may beprovided, for example, by the application of an ink or other coloringagent to the portions of the spacing layer adjacent the sample-receivingchamber. A colored section 23 of layer 14 is pictured in FIG. 2. Thechamber cover 20 is then provided as a transparent or translucentmaterial that allows the user to view the chamber and the adjacentspacing layer. As sample fluid enters from the edge of the test strip,the user is able to observe its progress as it moves by capillary actiontoward the vent opening. This type of feature is further described inU.S. Pat. No. 5,997,817, issued to Crismore et al. on Dec. 7, 1999, andis hereby incorporated by reference.

Capillary

The sample-receiving chamber formed by the base substrate, spacing layerand chamber cover essentially comprises several sections into which thesample fluid will travel. A first, entry section 48 extends from theedge opening to the area of the measuring electrode system. A second,test section 50 extends through the area of the electrode system. Athird section 52 extends from the measuring electrode system to the ventopening. It will be appreciated that the testing of the sample fluidoccurs in the area of the electrode system in the test section. However,the sample fluid will also fill the other sections of the chamber in thecourse of filling the test strip.

Dimensions.

The height and width of the sample-receiving chamber are selected basedupon a variety of considerations, including the fluid being tested andthe analyte at issue. For example, the chamber dimensions are preferablysized to promote capillary flow of the test fluid into the chamber.Preferred chamber heights for use with blood, for example, are fromabout 50 μm to about 200 μm, and most preferably from 120 to 180 μm. Ina preferred embodiment, the chamber height is about 150 μm. The width ofthe chamber may similarly be selected to match a desired sample fluidand analyte. For example, the chamber should be sufficiently wide toexpose a desired amount of the working and counter electrodes, andshould be narrow enough to avoid the requirement of an undue amount ofsample fluid for testing. The width of the sample-receiving chamber andthe width of the working electrode define the area of the workingelectrode. The area represents a further design consideration as itrelates to signal amplitude and instrumentation design.

Volume.

The sample-receiving chamber is preferably provided as having a minimalvolume, in order to reduce the amount of sample fluid needed forconducting a test. The overall sample-receiving chamber, including allof the three sections extending from the edge opening to the ventopening, has a total volume that can be considered to be a factor of thearea of the chamber from the edge to the vent, and the height of thechamber from the base substrate to the chamber cover 20. However, the“net chamber volume” comprises the volume of sample fluid required tofill this space. The net chamber volume of the sample-receiving chamberwill be the equivalent of the total chamber volume less the volumeoccupied by the electrodes, the reagent, and perhaps other items such asa sorbent material, if included.

As previously indicated, the volume of the overall sample-receivingchamber is comprised of the volumes attributable to the three sectionsof the chamber. Each of the sections is generally sized to be as smallas practical for the operation of the test strip. However, there areconsiderations, and possibly other functions, that will impact on thesize of each section.

The chamber volumes are a factor of both height and area. The height isa result of the thickness of the spacing layer and the thickness of theadhesives used to secure the spacing layer to the other layers. Forexample, the base substrate and the chamber cover are attached toopposite sides of the spacing layer. One method of attachment is theheat or laser sealing of the materials. It is preferred, however, toattach these layers by the use of suitable adhesives, such asheat-sensitive or pressure-sensitive adhesives. In this approach, theheight of the sample-receiving chamber, i.e., the distance between thefacing surfaces of the bottom substrate and the chamber cover, will beimpacted by the thickness of the adhesive layers. As shown in FIG. 3,chamber 24 is bounded on its bottom side by reagent layer 33 and its topside by coating 21 of chamber cover 20. However, adhesive layers 46 and49 as well as spacing layer 14 define the total height of chamber 24.

Further, in a preferred embodiment, the reagent layer 33 extends betweenbase substrate 12 and spacing layer 14 and indeed extends the entirewidth of the test strip, as described below. The height of the chambermay therefore also be increased due to the presence of the reagent layerunderlying the spacing layer. In this embodiment, and if adhesive isemployed, it has been found that the adhesive may combine with the testreagent, at least to an extent that causes the adhesive to fill somewhatinto and around the reagent. The heights of the reagent and adhesivelayers therefore are not necessarily additive in the final test strip.Rather, the height of the resulting space between the base substrate andthe spacing layer is somewhat less than the combination of the heightsof the separate reagent and adhesive layers prior to lamination.

It has also been found that the combination of the adhesive and thereagent advantageously helps to create a seal along the edge of thesample-receiving chamber. This inhibits sample fluid from wicking intothe reagent material present in the space between the base substrate andthe spacing layer in the time frame necessary for performing a test.

The first entry section is available to receive the sample fluid anddirect it to the measuring electrodes. This section can be quite smallin size, and may comprise only a short segment of the chamber. Thelength of this section is preferably less than 1200 μm.

The second testing section includes the test or measuring electrodes,and is also sized to require a minimal volume of sample fluid. A primaryfactor controlling the size of this second section will be the type,number, size, signal strength, and configuration of the measuringelectrodes. The length of this section is preferably about 1260 μm. Apreferred volume is about 0.265 μL, based on a capillary height of 0.15mm, and a capillary width of 1.4 mm.

The sample fluid moves past the measuring electrodes and into the thirdsection. This provides assurance, and preferably allows for specificconfirmation, that the measuring electrodes are properly wetted. Thisconfirmation may be by visual observation by the user, or by automaticdetecting means. For example, dose sufficiency electrodes may be placedin this section to detect when the sample fluid has progressed into thissection to a point that the wetting of the measuring electrodes isassured. This can be used as a trigger for initiating the application ofthe potential to the electrodes. The length of this section ispreferably 50 to 500 μm, and more preferably 255 to 400 μm. The volumeis preferably 0.01 to 0.1 μL, and more preferably 0.05 to 0.08 μL.

In a preferred embodiment, the overall net chamber volume of thesample-receiving chamber is less than about 1 μL, and is more preferablyless than about 0.5 μl. Desirable ranges for the net chamber volume ofthe sample-receiving chamber include volumes from about 0.15 to about1.4 μL, more preferably from about 0.4 to about 0.7 μl.

Sorbent.

The sample chamber may be otherwise empty, which is preferred, or mayalternatively include a sorbent material. Suitable sorbent materialsinclude polyester, nylon, cellulose, and cellulose derivatives such asnitrocellulose. A sorbent material could be included to facilitate theuptake of the sample fluid by assisting in wicking the fluid into thechamber. The use of a sorbent material would also serve to furtherreduce the void volume of the sample-receiving chamber for reception ofthe sample fluid.

Fill Method.

The preferred method of filling the sample chamber is by capillaryaction. In addition, the filling of the test strip can be augmented byother means, such as applying a pressure on the sample fluid to push itinto the sample chamber, and/or creating a vacuum on the sample chamberto pull the sample fluid into the chamber.

Hydrophilic Coating.

For purposes of capillary filling of the sample-receiving chamber,various approaches are available to facilitate the movement of thesample fluid into the chamber. For example, any or all of the surfacesdefining the chamber may be selected or treated to improvehydrophilicity. Such treatment may comprise the use of known hydrophilicmaterials, application of a hydrophilic material onto the surface, ortreatment of the surfaces to increase hydrophilicity, as describedbelow. In addition, the reagent composition may be formulated to bereadily hydrated and to encourage filling of the sample-receivingchamber. As previously indicated, a sorbent may also be used.

Testing for Analyte

The electrochemical sensor is operated by applying a suitable potentialor series of potentials across the working and counter electrodes, andacross the dose sufficiency electrodes. When a mediator is used, themagnitude of the required potential across the working and counterelectrodes will be dependent on the redox mediator. Moreover, thepotential at the electrode where the analyte is electrolyzed istypically large enough to drive the electrochemical reaction to or nearcompletion, but the magnitude of the potential is preferably not largeenough to induce significant electrochemical reaction of interferants.For glucose, for example, an applied potential difference typically isbetween about +100 mV and about +550 mV when using a DC potential. Whenusing AC potentials these can be typically be 5 to 100 mV RMS.

A potential may be applied before or after the sample begins to enterthe sample-receiving chamber. However, a potential is preferably appliedafter the sample has entered the chamber, and more preferably after ithas been determined that there is a sufficient amount of sample in thesample-receiving chamber for conducting a test. The timing of theapplication of a potential may be triggered in a variety of fashions,including visual observation by the user, a time delay followingsampling of the fluid to the test strip, or upon electrical or otherautomated detection of a sufficient amount of sample fluid in thechamber. The visual and electrical alternatives also may act asredundant failsafes to assure proper operation of the device.Preferably, the test strip and system utilize separate detecting means,such as dose sufficiency electrodes, for determining when the fluidsample has sufficiently filled the chamber.

When a potential is applied and the sample fluid is in thesample-receiving chamber, an electrical current will flow between theworking electrode and the counter electrode. The current can be a resultof the electrolysis of the analyte in the sample fluid when a potentialof sufficient magnitude is applied. In this case electrochemicalreaction occurs via the redox mediator, generally as previouslydescribed. In the case where small amplitude potential is applied,particularly in the case of AC potentials, the current is produced notnecessarily by electrolysis, but by ionic motion and response of thedielectric in the sample chamber. Those skilled in the art willrecognize that there are many different reaction mechanisms that willachieve the same result.

Control Solution

A test may be applied to the test strip after dosing to confirm that acontrol solution, and even that the correct control solution, has beenadministered. The control solutions aid the user in confirming that theentire system is functioning within design specifications, and that thetest strips have not been stored improperly or otherwise mistreated.Acceptable strips will recover values within specified tolerance rangesfor the particular strip lot being tested. The tolerance ranges inquestion will be published for each strip lot on the container label.

Method of Making Strip

In a preferred embodiment, the sensor comprises a multi-layered,laminate test strip 10. As previously described, the laminate includes abase substrate 12, a spacing layer 14, and a covering layer 16. Thesecomponents may be assembled in various ways. For example, the componentsmay be assembled by use of adhesives, heat sealing, laser welding, and avariety of other suitable techniques appropriate for securing theadjacent materials. The test strips are preferably assembled in a largenumber on a single sheet or web, and the strips are thereafter separatedfor storage and use.

The laminate test strip may be assembled sequentially by successivelylaying down one layer at a time. Alternatively, the test strip can beprepared by assembling and processing individual components or layers,which are then laminated together to provide the functional test strip.In one preferred form, two or more basic components of the test stripare prepared simultaneously. Then in one or a series of assembly orlaminating steps, the basic components are combined to produce the teststrip, which may or may not require further processing. In a preferredembodiment, the test strip is assembled from three basic components: ametallized substrate preferably with a reagent layer coated on metallicelectrodes defined on the substrate, a spacing layer having a cavitypreformed therein, and one or more top or cover layers.

With such small dimensions for the sample-receiving chamber, thecharacteristics of the reagent layer can have a significant impact onthe operation of the test strip, particularly in view of hydration andmixing characteristics. The reproducibility of the quantity, location,thickness and other properties of the reagent layer is thereforeimportant. It is therefore desirable for the composition to includematerials which specifically enhance the physical characteristics, suchas the uniformity and flatness, of the applied layer.

In one particular aspect, the test strip includes a unique manner ofincorporating the reagent. The reagent is placed in the sample-receivingchamber at least on the working electrode, and preferably also on thecounter electrode. The reagent may be applied to the test strip in avariety of fashions as is well understood in the art. In a preferredembodiment, the reagent composition is applied as a thin coating overthe electrodes supported on the base substrate.

More particularly, the reagent is placed onto the base substrate in amanner that positions the reagent composition between the base substrateand the spacing layer. This manner of application helps to make thereagent layer more flat and uniform in thickness. In contrast, aprocedure of the prior art has been to first prepare the reaction wellor cavity, and to then fill the reagent into the well. However, this canresult in a more uneven reagent layer due to phenomena such as theformation of a meniscus at the perimeter of the well. This in turn cancause the reagent to have a different thickness adjacent to the sidewalls of the reaction well than in the interior portion, which can causeinconsistency in the filling of the chamber, prolonged dissolutionintervals, and inconsistent mixing of the reagent with the sample fluid,and the ultimate test results. By placing the reagent onto the basesubstrate before the spacing layer is added, there is no meniscus effectto disrupt the even layering of the reagent as it dries on the basesubstrate. In addition, this method of application facilitates the massproduction of the test strips.

Referring to the drawings, the test strip 10 is shown as including areagent layer 33 that extends between the bottom substrate 12 and thespacing layer 14. More particularly, the reagent forms a layer 33 whichcovers both the top surface of the bottom substrate 12 and theelectrodes 28. The reagent covers at least the working electrode, andpreferably also the counter electrode. In the most preferred embodiment,the reagent layer extends the full width of the test strip. The reagentlayer also preferably extends from the end edge to the dose sufficiencyelectrodes, and most preferably to the vent opening. The reagent layerthus extends under the spacing layer and is sandwiched between thespacing layer and the base substrate.

The reagent composition is applied to the bottom or base substrate inany suitable fashion that provides a desired and uniform layer whichwill ultimately extend under the spacing layer. The reagent ispreferably applied in a continuous coating directly onto the bottomsubstrate, and onto the electrodes received thereon. As describedhereafter, the reagent composition is most preferably applied in thecourse of producing a large quantity of test strips on a webbing ofmaterial. In this manner, the reagent may be applied in the way of acontinuous stripe of material that extends over a substrate roll that islater separated into individual test strips. The reagent composition isallowed to dry or otherwise set up and the spacing layer is appliedthereover.

In a related aspect, a preferred manner of securing the spacing layer tothe bottom substrate is the use of an adhesive. In addition to securingthe layers together, it has been found that the adhesive willsufficiently engage with the reagent composition as to help to seal thespace between the bottom substrate and the spacing layer. The adhesivespreferably placed on the spacing layer, which is laminated onto the basesubstrate. The adhesive thereby contacts the portion of the reagentwhich extends under the spacing layer.

Although the spacing layer of the illustrated embodiment is formed fromMelinex® material with adhesives on both sides thereof, it is alsopossible to form spacing layer 14 as a continuous adhesive material,such as a double-sided tape. For example, a 5 to 6 millimeter thickARCare Adhesive could be used in lieu of spacing layer 14.

In a further aspect, a preferred embodiment is described in which theanalyte is glucose. In the case of glucose, the active components of thereagent composition will typically include an oxidoreductase, such as anenzyme for glucose; optionally a co-enzyme or co-factor; and a redoxmediator. These components are typically dissolved or suspended in amatrix. The liquid test sample hydrates or dissolves the matrix, and theanalyte diffuses through the matrix to react with one or more of theactive components. Typically, the enzyme oxidizes the glucose in thetest sample to gluconolactone and/or gluconic acid. The mediator, inturn, reacts with or oxidizes the reduced enzyme, and consequently themediator is reduced in the process. The reduced mediator can be detectedat one of the electrodes on the test strip.

In a specific example of an oxidation/reduction reaction scheme usefulfor detecting glucose in human blood, a test sample containing glucosereacts with an enzyme such as Glucose-Di-Oxidoreductase (Gluc-Dor), andoptionally a co-enzyme or cofactor such as pyrrolo-quinoline-quinone(PQQ), in the presence of a redox mediator. The mediator may include,for example, benzoquinone, transition metal complexes, e.g., potassiumferricyanide, osmium derivatives (e.g., osmium bipyridyl complexes suchas described in WO 98/35225) and nitrosoanaline derivatives (see U.S.Pat. No. 5,286,362). This produces the oxidized form of the analyte,gluconolactone, and the reduced form of the redox mediator. The mediatorthereafter shuttles the redox equivalent of mediator product, thereduced mediator, to the electrode surface by diffusion. There themediator is oxidized quantitatively at a defined anodic potential, andthe resulting current is related to the apparent glucose concentration.

A representation of the reaction sequences for this reaction systemusing a nitrosoaniline derivative is provided below in Equation 1.

As shown, the nitrosoaniline derivative,o-methoxy-[N,N-bis-(2-hydroxyethyl)]-p-nitrosoaniline, initially existsas a mixture of two isomers, or tautomers, in equilibrium with eachother. Reaction of Gluc-Dor with glucose in the test sample yieldsgluconolactone and the reduced form of Gluc-Dor (Gluc-Dor·2H⁺). Thereduced form of Gluc-Dor (Gluc-Dor·2H⁺) reacts rapidly with thenitrosoaniline derivative, which is reduced and which regeneratesGluc-Dor. The reduced nitrosoaniline derivative then undergoeshydrolysis to form quinonediimine (QD). In a second enzymatic, redoxreaction, Gluc-Dor reacts with glucose to yield another molecule ofGluc-Dor·2H⁺ and gluconolactone. The Gluc-Dor·2H⁺ reacts with (isoxidized by) quinonediimine to regenerate Gluc-Dor, and produces aphenylene diamine derivative (PD). PD is then oxidized at the workingelectrode to produce a current related to glucose concentration.Additionally, at the counter electrode QD can be reduced to PD.

Adjuvants.

With such small dimensions for the sample-receiving chamber, thecharacteristics of the reagent layer can have a significant impact onthe operation of the test strip, particularly in view of hydration andmixing characteristics. The control and reproducibility of the quantity,location, width, thickness, and other properties of the reagent layerbecome more important as the chamber volume decreases and test timediminishes. It is therefore desirable for the composition to includematerials that specifically enhance the physical characteristics, suchas the uniformity and flatness, of the applied layer. Additionally, themethod of application can impact the physical characteristics, control,and reproducibility of the reagent layer.

The reagent composition can therefore also include a variety ofadjuvants to enhance the reagent properties or characteristics. Forexample, the composition may include adjunct materials to facilitate theplacement of the reagent composition onto the test strip and to improveits adherence to the strip. The composition can also include materialsto increase its rate of hydration and/or increase its influence on thecapillary action to fill the chamber with the test sample. Examples ofaddjunct materials to be used with the reagent composition includethickeners, viscosity modulators, film formers, stabilizers, buffers,detergents, gelling agents, fillers, film opening agents, coloringagents, and agents endowing thixotropy.

The adjuvant materials or components can impact the application,reproducibility and physical properties of the reagent layer. Theadjunct materials can include one or more of the following:

Thickeners may include, for example, (1) starches, gums (e.g., pectin,guar gum, locust bean (carob seed) gum, konjac gum, xanthan gum,alginates, and agar), casein, gelatin, and phycocolloids; (2) celluloseand semi-synthetic cellulose derivatives (carboxymethyl-cellulose,methyl cellulose, hydroxymethylcellulose, hydroxyethylcellulose,methylhydroxyethylcellulose); (3) polyvinyl alcohol andcarboxy-vinylates; and (4) bentonite, silicates, and colloidal silica.Preferred thickeners include a combination of a xanthan gum sold underthe trade name Keltrol F by CP Kelco US, Inc., and carboxylmethylcellulose sold under the trade name AQUALON® CMC 7F PH by Hercules Inc.,Aqualon Division.

Film forming and thixotropic agents useful in the reagent compositioninclude polymers and silica. Preferred thixotropic agents include silicasold under the trade name Kieselsaure Sipernate FK 320 DS by Degussa AG.Preferred film forming agents include polyvinylpyrrolidone, sold underthe trademark polyvinylpyrrolidon Kollidon 25, by BASF, and polyvinylpropionate dispersion.

Stabilizers for the enzyme in the reagent can be selected fromsacchhrides and mono-or di-fatty acid salts. Preferred stabilizersinclude trehalose sold under the trade name D-(+)-Trehalose dihydrate bySigma Chemical Co. and sodium succinate.

Detergents can be selected from water-soluble soaps, as well aswater-soluble synthetic surface-active compounds such as alkali, earthalkali or optionally substituted ammonium salts of higher fatty acids,e.g., oleic or stearic acid, mixtures of natural fatty acids, forexample, from coconut or tallow oil, fatty sulphates, esters ofsulphonic acids, salts of alkyl sulphonic acids taurine salts of fattyacids, fatty acid amides, and ester amides. Preferred detergents for thepresent invention include an ester amide, n-octanoyl-N-methylglucamide,sold under the trade name Mega-8 by Dojindo Molecular Technologies,Inc., and a fatty acid salt, N-methyl oleyl taurate sodium salt, soldunder the trade name Geropon T77 by Rhodia HPCII (Home, Personal Careand Industrial Ingredients).

It should be understood that one or more of the specific additives abovedescribed can exhibit additional properties and consequently could becategorized in one or more of the classes above noted.

Mediator.

A mediator for use in the reagent composition can be selected as anychemical species (generally electroactive) which can participate in areaction scheme involving an enzyme, an analyte, and optionally acofactor, and reaction products thereof, to produce a detectableelectroactive reaction product. Typically, participation of the mediatorin the reaction involves a change in its oxidation state (e.g., areduction), upon interaction with any one of the analyte, the enzyme, ora cofactor, or a species that is a reaction product of one of these(e.g., a cofactor reacted to a different oxidation state). A variety ofmediators exhibit suitable electrochemical behavior. A mediator canpreferably also be stable in its oxidized form, may optionally exhibitreversible redox electrochemistry, can preferably exhibit goodsolubility in aqueous solutions, and preferably reacts rapidly toproduce an electroactive reaction product. Examples of suitablemediators include benzoquinone, meldola blue, other transition metalcomplexes, potassium ferricyanide, osmium derivatives (see WO 98/35225)and nitrosoanaline-based mediators (see U.S. Pat. No. 5,286,362). In apreferred embodiment, the reagent composition utilizes anitrosoaniline-based chemistry.

Preferred mediators includeN-(2-hydroxyethyl)-N′-p-nitrosophenyl-piperazine,N,N-bis-(2-hydroxyethyl)-p-nitrosoaniline,o-methoxy-[N,N-bis-(2-hydroxyethyl)]-p-nitrosoaniline,p-hydroxynitrosobenzene, N-methyl-N′-(4-nitrosophenyl)-piperazine,p-quinone dioxime, N,N-dimethyl-p-nitrosoaniline,N,N-diethyl-p-nitrosoaniline, N-(4-nitrosophenyl)-morpholine,N-benzyl-N-(5′-carboxypentyl)-p-nitrosoaniline,N,N-dimethyl-4-nitroso-1-naphthylamine,N,N,3-trimethyl-4-nitrosoaniline, N-(2-hydroxyethyl)-5-nitrosoindoline,N,N-bis-(2-hydroxyethyl)-3-chloro-4-nitrosoaniline,2,4-dimethoxy-nitrosobenzene, N,N-bis-(2-methoxyethyl)-4-nitrosoaniline,3-methoxy-4-nitrosophenol,N-(2-hydroxyethyl)-6-nitroso-1,2,3,4-tetrahydroquinoline,N,N-dimethyl-3-chloro-4-nitrosoaniline,N,N-bis-(2-hydroxyethyl)-3-fluoro-4-nitrosoaniline,N,N-bis-(2-hydroxyethyl)-3-methylthio-4-nitrosoaniline,N-(2-hydroxyethyl)-N-(2-(2-methoxyethoxy)-ethyl)-4-nitrosoaniline,N-(2-hydroxyethyl)-N-(3-methoxy-2-hydroxy-1-propyl)-4-nitrosoaniline,N-(2-hydroxyethyl)-N-(3-(2-hydroxyethoxy)-2-hydroxy-1-propyl)-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)-ethyl)-4-nitrosoaniline.Particularly preferred mediators according to the present inventioninclude N,N-bis-(2-hydroxyethyl)-p-nitrosoaniline,o-methoxy-[N,N-bis-(2-hydroxyethyl)]-p-nitrosoaniline, andN-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)-ethyl)-4-nitrosoaniline.

An exemplary reagent composition is listed below in Table I. TABLE IComponents Function Amount Abs. % solids w/w. Note. Keltrol F Thickener 11.60 g 0.24% Carboxy methylcellulose Thickener  27.24 g 0.57%Kieselsäure Sipernat 320 DS Film Opener  97.01 g 2.01%Polyvinylpyrrolidine PVP K25 Film Former  89.33 g 1.85% Propiofan FilmFormer 257.09 5.34% GlucDOR Apo-Enzyme  19.127 g 0.40% 0.673 MU/gpyrrolo-quinoline quinine Co-Factor  0.5329 g 0.01% (PQQ) Na-SuccinateStabilizer  23.23 g 0.48% Trehalose Stabilizer   23.6 g 40.49%  KH₂PO₄Buffer  12.02 g 0.39% K₂HPO₄ × 3 H₂O Buffer  43.43 g 0.90%Nitrosoaniline Mediator  41.26 g 0.86% Mega 8 Detergent  13.23 g 0.27%Geropon T77 Detergent  1.405 g 0.03% KOH 5N Adjust Buffer  36.47 g 0.76%Water total 4114.52 g Sum 4817.80 g Solids 14.6%Mixing.

The components of the reagent composition are admixed with water toprovide a homogeneous, viscous suspension. The order of addition is notcritical for the invention. A sufficient amount of the buffer solutionis added to maintain the reagent composition at a pH of about 7.Typically the selected components are pre-mixed with water to provide avariety of stock solutions that can be combined to yield the finalreagent composition. For example, a buffer solution can be prepared bycombining the phosphate salts and, optionally, the sodium succinate.Other stock solutions include: the thickening agents, i.e., Keltrol Fand the carboxymethyl cellulose; the surfactants, i.e., Geropon T77 andMega 8; the enzyme and co-enzyme or cofactor; and the mediator.

The following provides an example of the preparation of a reagentcomposition. The reagent composition can be prepared by first preparingthe following stock solutions: Buffer Solution pH 6.9 to 7.1 Amount (gm)H₂O 1214.62 KH₂PO₄ 18.27 K₂HPO₄ 43.43 Na succinate 23.23

Keltrol F Solution Amount (gm) H₂O 287.06 Buffer Solution 101.35 KeltrolF 11.60

Carboxymethylcellulose (CMC) Solution Amount (gm) H₂O 1334.76 Na CMC¹27.24¹Na CMC is a sodium salt of carboxymethyl cellulose sold by HerculesInc.,

Aqualon Division Silica Suspension Amount (gm) H₂O 722.99 Sipernat 320¹¹Kieselsäure Sipernat 320 DS (Silica) sold by Degussa AG.

Polyvinylpyrrolidone (PVP) Solution Amount (gm) Buffer Solution 226.03Mega 8¹ 13.23 Geropon T77² 1.405 PVP³ 89.33¹Mega 8 is n-octanoyl-N-methylglucamide sold by Dojindo MolecularTechnologies Inc.²Geropon T77 is N-methyl oleyl taurate sodium salt sold by Rhodia HPCII.³PVP is Polyvinylpyrrolidone K25 sold by BASF.

Trehalose Solution¹ Amount (gm) H₂O 36.4 Trehalose 23.6¹This trehalose solution is used only in preparing the “Enzyme Solution”listed below.

PQQ Solution Amount (gm) Buffer Solution 1^(st) 101.59 addition PQQ0.533 Buffer Solution 2^(nd) 30.0 addition

Enzyme Solution Amount (gm) PQQ Solution 132.12 Gluc-Dor 19.13 (673 U/mgLy) Trehalose Solution 58.75

Mediator Solution Amount (gm) Buffer Solution 782.27 Mediator 41.26 5 NKOH 36.47

The buffer solution, Keltrol F solution, CMC solution, and the Silicasuspension were prepared a day before. These solutions can then becombined as listed below to prepare the reagent composition.

Final Reagent Composition Thickener I 331.51 g (Keltrol F solution)Thickener II (CMC 1262.9 g Solution) PVP Solution 315.05 g Silicasuspension  762.3 g Propiofan solution 257.09 g Mediator Solution 855.84g Enzyme Solution 196.65 g 5 N KOH as required to achieve final pH of6.9 to 7.1 Water (bidistilled) 518.69 gFor this reagent prior to coating, the final pH was 6.96 and did notneed adjustment with 5N KOH solution. The measured viscosity was 111mPas, which is in the correct range for coating of 105 to 115 mPas.

FIGS. 5 and 5A present a flow chart illustrating a preferred process 100for preparing a test strip useful in accordance with the presentinvention. Process 100 begins in the central process line 101 at stage102 with selection of a film material for the base layer or basesubstrate. In a preferred embodiment, the film is provided as acontinuous roll having a width and length suitable for fabricating alarge number of test strips. In subsequent finishing steps, theprocessed film can be subdivided to provide a single strip or web havinga width that approximates the length of the test strip and includes aseries of test strips, or can be die cut to provide individual testsensors.

From stage 102, the film proceeds to stage 104 where it is pretreated toreceive a metal coating and coated with the metal in one continuousprocess. The pretreatment 164 (discussed below) can be used to clean ormodify the surface to provide a uniform coating thickness and betteradhesion of the subsequent metallized layer 166 (discussed below). Thepretreatment can include subjecting the film to corona discharge orArgon plasma. Immediately after this pre-treatment, a uniform conductivecoating is applied to the film as shown at 106. Alternatively, suitablesubstrates with metal coatings can be obtained commercially.

The metallic layer may contain pure metals, oxides, alloys, or othermaterials, which are metallic conductors. Examples of metals ormetallic-like conductors include: aluminum, carbon (such as graphite),cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium,mercury (as an amalgam), nickel, niobium, osmium, palladium, platinum,rhenium, rhodium, selenium, silicon (such as highly dopedpolycrystalline silicon), silver, tantalum, tin, titanium, tungsten,uranium, vanadium, zinc, zirconium, mixtures thereof, and alloys orsolid solutions of these materials. Preferably, the materials areselected to be essentially unreactive to biological systems; suchmaterials include: gold, platinum, palladium, iridium, or alloys ofthese metals. The metallic layer may be any desired thickness.

The conductive coating is preferably a metal layer that is applied by avariety of methods, including but not limited to sputtering, physicalvapor deposition (PVD), plasma assisted vapor deposition (PAVD),chemical vapor deposition (CVD), electron beam physical vapor deposition(EBPVD), and/or metal-organic chemical vapor deposition (MOCVD). Vapordeposition is typically performed under vacuum. These techniques arewell known in the art and can be used to selectively provide a uniformlythin coating of metal onto a substrate. The resulting metallized filmcan be inspected to ensure that the metal coating is uniform and free ofmaterial defects.

The roll of metallized film next encounters stage 108 where it issubdivided and/or sized to provide webs having a width that approximatesthe final length of an individual test strip. The slicing can beaccomplished using fixed-knife slitting equipment well-known in the art.

A single web proceeds to stage 110 for patterning the electrodes,traces, and contacts or pads. At this stage, the electrodes, traces, andcontact pads are formed by removing metal from the surface of the webstrip. The excess metal can be removed using a variety of techniqueswell known in the art. At this stage, one or more indexing orregistration marks can be formed either on a first edge proximate to theelectrodes, the opposite second edge proximate to the electrode pad, onboth edges or anywhere in between. The indexing marks, particularlythose at an edge, can be used in subsequent operations to align layeredcomponents prefabricated in separate operations.

In a preferred method, the metal is laser ablated to eliminate undesiredportions of the metal and leave the desired electrical components. Inaccordance with this method, selected areas are laser etchedsimultaneously, in a “broad field”, as opposed to using linear movementof a focused laser beam. This broad field laser ablation method providesa precise metal pattern rapidly and at reduced cost as compared to otherapproaches. Corona treatment of the patterned substrate is thenconducted at stage 111.

The patterned web continues on to stage 112, where a reagent layer isdeposited onto the electrodes. In a preferred embodiment, the reagentlayer is deposited as a continuous elongate stripe extending adjacent orclose to the first edge, and overlying the measuring electrodes formedon the patterned web. As previously noted, the reagent is consequentlylocated across the full width of the test strip, including the arealaterally outside of the sample-receiving chamber and between the basesubstrate and the spacing layer. Also as noted, this will facilitate thedrying of the reagent without discontinuities, edge effects, or othervariances that would detract from providing a thin, flat, uniformreagent layer within the sample-receiving chamber. The reagent includesa combination of components, and is formulated to dry rapidly withminimal or no running after deposition, typically by manipulating thethixotropy of the coated reagent film.

This stripe may be applied in any suitable fashion which provides thedesired extent and uniformity of thickness, precision of the stripeedge, homogeneity, and the like. Preferred methods are capable ofapplying the desired coating at relatively high speed and high batchsize. Suitable methods of application are well known in the art andtherefore are not detailed herein.

Preparing the Spacing Layer

Referring now to process line 114, a process flow for preparing thespacing layer is illustrated. Beginning at stage 116, a material isselected to prepare a spacing layer for laminating on top of the reagentcoated web prepared at stage 112. The base film for the substrate can beselected from a variety of materials. The spacing layer material,similar to the base layer, can be provided as an elongate roll which canbe conveniently processed rapidly and with high efficiency. Preferredmaterials include a polyester film sold under the trade name MELINEX® byDuPont. Other materials suitable for use in the present invention couldinclude PEN. The spacing layer material has a thickness specificallyselected to provide a desired chamber depth (or height) in each of thetest strips when combined with the thicknesses of any joining layersthat are used to laminate the spacer to the other strip components. Inpreferred embodiments, the spacing layer is selected to have a thicknessbetween about 75 μm and about 150 μm, more preferably from about 100 μmto about 125 μm. As noted above, the spacing layer can be formed of adouble-sided adhesive.

The spacing layer is preferably formed as a continuous film having aseries of gaps that will align with the electrodes on the bottomsubstrate webbing. The manner of joining the spacing layer and bottomsubstrate will impact the method for preparing the spacing layer. Forexample, if the spacing layer is to be heat welded to the bottomsubstrate, then the spacing layer may simply be die cut to provide theappropriately spaced chamber gaps. However, a preferred method is theuse of thin, non-interfering adhesives that join the adjacent layers. Inaccordance with this preferred method, a spacing layer is prepared forcombination with the previously described substrate webbing as set forthhereafter.

The spacing layer film is prepared having the desired width forcombination with the remainder of the test strip components. The spacinglayer film may include an opaque portion, e.g., a section 23 of it isprinted blue or another color for use in visualizing thesample-receiving chamber, as described elsewhere. The spacing layer filmis laminated on the bottom side with a combination adhesive and releaseliner, and on the top side with a similar combination adhesive andliner.

At stage 118, two transfer adhesives are laminated to the spacing layermaterial: the first transfer adhesive is laminated to the top surface ofthe spacing layer, and the second transfer adhesive is laminated to thebottom surface of the spacing layer. Preferably, the transfer adhesivesare the same adhesive; however, in alternative embodiments, the firstand second transfer adhesives can be different from each other. Inpreferred embodiments, the transfer adhesives are selected from commonlyused and known adhesives, including pressure sensitive adhesives.Preferred adhesives exhibit sufficient hydrophobicity to prevent orinhibit the test sample in the chamber from wicking out between thespacing layer and the reagent layer or base substrate. An example of asuitable sensitive adhesive is ARCare 90132 from Adhesives Research Inc.The adhesives are provided with a release liner to prevent prematureadhesion of the spacing layer during processing. The release liners aredisposed on the exterior surface of the first and second transferadhesives, facing outward from the spacing layer material.

The spacing layer with the adhesive release liners on the top and bottomsurfaces progresses to stage 120. At stage 120, the cavity which willform the sample-receiving chamber is punched in the spacing layer. Inone embodiment, the cavity is punched using a “kiss cut” method. Thekiss cut method cuts through the upper release liner, the upperadhesive, the spacing layer, and the lower adhesive, but not through thelower release liner. In subsequent operations, simply removing the lowerrelease liner will then remove the punched out portions of the loweradhesive, the spacing layer, the upper adhesive, and the upper releaseliner from the punched spacing layer. In other embodiments, the cavitycan be punched through with a hollow die. The hollow die completelypunches or cuts through the spacing layer, the two adhesives, and thetwo release liners, with the punched out portion subsequently removed inthe hollow die. The spacing or pitch between each cavity is determinedand accurately controlled to allow accurate mating of the punchedspacing layer over the electrodes using one or both of the indexingmarks patterned in the reagent-coated web.

At stage 122, the lower release liner on the spacing layer is removed,taking with it the kiss cut portions and exposing the adhesive on theunderside surface of the spacing layer. Proceeding on to stage 124 inprocess line 101, the spacing layer is laminated over the reagent-coatedweb using one or more of the indexing marks previously patterned on theweb to correctly align each cavity formed in the punched spacing layerdirectly on top of an electrode set to provide a web-spacing layerlaminate. At stage 126 in the central process line 101, the upperrelease liner covering the upper adhesive on the web-spacing layerlaminate is removed in preparation for attaching the cover layer.

Laminating on the Cover Portions

At stage 128, a material for a body cover is introduced into theprocess. In preferred examples, the material is a flexible polymericmaterial and may be selected, for example, MELINEX 454 or MELINEX 339from du Pont. The material for the body cover is sized to have a widthsufficient to overlay at least a portion of the electrode traces in thesample-receiving chamber of the test strip.

Referring now to process line 130, beginning at stage 131, a filmmaterial is selected to provide a chamber cover over the cavity,reagent, and measuring electrodes on the web-spacing layer laminate. Inpreferred embodiments, the body cover material is provided as a clearpoly(ethylene-terephthalate) (PET) or poly(ethylene-naphthalate) (PEN)film having a thickness between about 100 μm and about 200 μm. Thecoating may preferably include a release liner, which can be removedimmediately prior to laminating over the web-spacing layer. The chambercover is preferably made from a hydrophilic material or the bottomsurface of the chamber cover may be treated or coated to make ithydrophilic as indicated at 134. At stage 138, the film material can besized to a desired width sufficient to form the chamber cover to overlaythe cavity and electrodes.

Proceeding to stage 140, the body cover from stage 128 and the chambercover from stage 138 are laminated to the web-spacing layer laminate. Inpreferred embodiments, the body cover and chamber cover aresimultaneously laminated with the web-spacing layer laminate. The bodycover is positioned over a portion of the electrode traces proximate tothe electrodes formed on the base substrate. The chamber cover ispositioned over the cavity, reagent, and measuring electrodes on theweb-spacing layer laminate. The body cover and chamber cover areseparated by a gap to form a vent 34 at the interior end of the cavityformed in the test strip.

As described, the chamber cover is placed near the edge of the strip tooverlie the cut out portion of the spacing layer, leaving the innermostportion of the cut out uncovered. As just described, this chamber coverpreferably includes a hydrophilic underside to promote the wicking offluid into the reagent chamber. The chamber cover is spaced slightlyfrom the body cover to form a gap which thereby communicates with thesample-receiving chamber and serves as a vent opening for air to escapeas fluid enters the chamber, as described above.

The opacity of the spacing layer and the transparency of the chambercover cooperate to allow a user of the final test strip to better viewthe progress of a test. As constructed, the bottom substrate or reagentlayer coated thereon is visible through the cut out in the spacing layerand through the transparent chamber cover. The bottom substrate and/orreagent has a light color, e.g., bright yellow, which contrasts with theopaque coloring of the spacing layer. Therefore, the progress of a fluidthrough the capillary channel can be easily monitored by the personusing the test. Further, since the slot 34 is configured to behydrophobic on the body cover side and hydrophilic on the chamber coverside, fluid will abruptly stop when it reaches the slot, thus presentinga sharply defined fill-line which in turn provides a clear indication tothe user that sufficient fluid sample has been received into thechamber.

Separating the Test Strips

From stage 140, the finish processing steps for the fabrication of teststrips are performed. At stage 141, a profile cut is made across the endof the web of test strips 10. Typically, the profile cut is made in twosteps. First, the web is sheared by a cutting blade moving across theweb, thereby forming a straight edge; then the web is die-cut to producethe desired profiled shape (a taper for the illustrated embodiment) ofthe dosing end 36 of the test strips.

At stage 142, any graphics or logos are printed on the test strips 10.

At stage 143, a decision is made whether to manufacture a single,die-cut test strip similar to the single test strip 10 above discussed.If so, then the multi-layered laminate from stage 143 proceeds to stage144 to be die cut into single test strips.

Alternatively, the multi-layered laminate from stage 143 proceeds tostage 148, where it is kiss cut to define individual test strips and toperforate or weaken the boundaries between adjacent test strips on theribbon. Additionally, at stage 149 the ends of the test strips are diecut along the laminated ribbon. One end of the web is cut to form thefluid receiving end of the test sensor with a Y-shaped opening leadinginto the cavity. The test strips may be divided into cards comprising anumber, e.g., 25, of strips which are only fence cut and then folded tobe stacked in a vial or dispenser.

Proceeding out of either stage 144 or 149, the processed strips orribbon of strips are inspected and ultimately packaged for use by theconsumer at stage 146 or 150, respectively.

FIGS. 6-16 illustrate in greater detail some of the components and/orprocess stages previously described with respect to FIGS. 5 and 5A. FIG.6 illustrates a perspective view of one embodiment of a base film foruse in forming the test strip. Base film 160 is preferably provided as aflexible film or web material that is rolled onto one or more rollers162 with processes 164, 166 proceeding on the material between therollers.

The pretreated upper surface of the film is metallized using asputtering, PVD, CVD, EBPVD, MOCVD or another suitable process,illustrated by reference number 166 and described more fully above, todeposit a uniform coating of a metal or metal alloy. The processes canuse a single or multiple target source for the metallic layer. Themetallized film 168 can then be sectioned or subdivided into a pluralityof metallized films, e.g., 170 a, 170 b, and 170 c, by cutting orslicing the film as illustrated by reference number 172. Each separatedroll of the conductive, metallized film 170 can then be rolled upon asingle core or upon a plurality of different cores as preferablydesired.

The electrical components are formed from the conductive film, as shownin one embodiment in FIG. 7. The metallic surface of film 170 is treatedto remove any metallic component that is not desired to form theelectrodes, traces, contact pads, or other intended features. Thisprocess can be precisely controlled using laser ablation or othertechnology. The process provides a plurality of sets of electrodes 182,traces 184, and contact pads 186. The process can also provide aplurality of indexing or registration marks 176 along a first edge 178and/or similar registration marks 177 along the opposite edge 180. Asshown in FIG. 7, repeating features of the electrode pattern formregistration markings 177. Preferably, each set of electrodes and/orcontacts is associated with at least one index or registration mark, 176and 177, respectively.

FIG. 8 illustrates a portion of a reagent-coated web 188. The reagentcomposition is deposited on the surface of the flexible web material.The reagent layer 190 is deposited using a variety of coating methodsincluding curtain coating, hot melt coating, rotary screen coating,doctor blade or air knife coating, Meyer bar coating, and reverse rollcoating techniques. Preferably, the reagent layer is deposited on theflexible web as a wet composition at a thickness of between about 50 μmand about 100 μm, more preferably, between about 60 μm and about 90 μm.Web 188 can be provided by coating a uniformly thin layer of reagent 190directly on top of electrode sets 182 and along the length of web 188 asa continuous narrow band 192. In preferred embodiments, the narrow band192 has a width of between about 5 mm and 9 mm and a dry thickness ofbetween about 2 μm and about 10 μm. As depicted in FIG. 8, the reagentlayer 190 is translucent.

FIG. 9 is an exploded view of a spacing layer assembly 194, which can beassembled in accordance with the present invention. Spacing layerassembly 194 comprises a spacing layer 196 preferably formed of apolymeric material. Spacing layer 196 includes a band or section 197that is colored (corresponding to section 23, FIG. 2). In themanufacturing process, spacing layer 196 is provided in a roll 198 andis then overcoated with adhesives on top and bottom.

The top adhesive is provided in a roll 200 which further comprises a topor “tight” release liner 202, which is adapted to withstand furtherprocessing, an adhesive 204, and a lower or “easy” release liner 206.Preferred adhesives 204 for use in the present invention include apressure-sensitive adhesive sold under the trade name ARCare 90132 byAdhesives Research Inc. During assembly, bottom release liner 206 isremoved and the resulting adhesive 208 having the top release liner 202still present is adhered to spacing layer 196 as indicated in the top ofFIG. 9.

Similarly, the bottom adhesive is provided in a roll 210 which furthercomprises a top or “tight” release liner 212, which is adapted towithstand further processing, an adhesive 214, and a lower or “easy”release liner 216. Preferred adhesives 214 for use in the presentinvention include a pressure-sensitive adhesive sold under the tradename ARCare 90132 by Adhesives Research Inc. During assembly, bottomrelease liner 216 is removed and the resulting adhesive 218 having itstop release liner 212 facing away from spacing layer 196 is adhered tospacing layer 196 as indicated in FIG. 9. It should be understood thatadhesive 204 can be the same or different from adhesive 214.

FIG. 10 illustrates spacing layer 196 that has been die cut to formpre-capillaries 220 a, 220 b, 220 c, etc., and is ready to be laminatedto a web of base substrate material 188 as described with reference toFIG. 8. Pre-capillaries 220 can be formed using a “kiss-cut” techniquein which a die cuts through the top release liner 202, adhesive 204,spacing layer 196, and adhesive 214, but not release liner 212, which,as noted above, faces away from spacing layer 196. Release liner 212 isthen removed along with portions of the top release liner 202, adhesive204, spacing layer 196, and adhesive 214 that had been cut through.These portions that are cut through comprise “capillary trim,” i.e., asandwich of layers shaped like pre-capillaries 220. This “trim” isremoved along with release liner 212, leaving the cavities 220 devoid ofany material. As release liner 212 is removed, it can be inspected toensure that it always contains the capillary trim just described. Theresulting series of cavities 220 are spaced from each other a desireddistance selected to position each one of the channels of the series ofchannels 220 directly over a measuring electrode set in the test strip.The spacing layer 196 having its lower adhesive exposed can then bealigned with web 188 by means of indexing marks 176 and laminatedthereto. Each capillary channel of the series of channels 220 overlaysone set of measuring electrodes 182.

FIG. 11 illustrates an assembly 230 formed by the lamination of spacinglayer 196 to web 188. In FIG. 11, the upper release liner 202 has beenremoved from the top adhesive 208, which makes assembly 230 ready forassembly of additional material thereto. As shown in FIG. 12, a web 240of chamber covering layer material and a web 234 of body coveringmaterial are aligned over the exposed upper adhesive 208 of assembly 230and are ready to be adhered thereto. As depicted in FIG. 12, chambercovering layer 240 is clear and includes a hydrophilic coating (seecoating 21, FIG. 2) on at least the side that faces cavities 220. Thisfacilitates wicking or transport of the liquid sample into thesample-receiving chamber and over the electrodes and reagent layer. Bodycover 234 is opaque, is colored as shown, and is preferably hydrophobic.Covering layer 240 and body cover 234 can be provided on reels likethose described above with reference to FIG. 9.

Preferably, chamber covering material 240 is slightly thinner than bodycovering material 234. After the chamber covering material 240 and bodycovering material 234 are laminated to the other layers (describedbelow), the assembly is rewound to await the final processing steps. Ifbody covering material 234 is thicker than chamber covering material240, then body covering material 234 will absorb more of the pressure orforce imparted to the web as it is rewound and stored. Thus, if anyadhesive squeezes out of the web as it is rewound, the adhesive willsqueeze out around the body covering material 234 and not the chambercovering material 240. Advantageously, the thinner chamber cover thusreduces the possibility of the adhesive squeezing out from under itduring roll processing and entering the capillary zone where it coulddegrade or destroy the test strips ultimately produced. Additionallywhen assembly 260 (discussed below) is re-rolled onto a core, thepressure exerted on the chamber covering material 240 is less than thatexerted on body covering material 234 which minimizes the possibility ofdamage during processing to the capillary chamber.

Assembly 260 shown in FIG. 13 is produced by laminating webs 234 and 240to the assembly 230 shown in FIG. 12 and then trimming the end of theweb to form dosing edge 250. Dosing edge 250 is preferably formed by ashear cut in which the cutting blade moves across the end of the web asindicated by arrow 252. By contrast, it is more difficult to use a diepunching technique without damaging the capillaries. The shear cut alongdosing edge 250 also cuts away a portion of the pre-capillaries 220 anddefines the final volume of capillaries 222. Capillaries 222 preferablyinclude a flared or Y-shaped opening as shown. Preferably, a gap 262 isformed between the chamber covering web and the body covering web andthis gap will ultimately provide a vent opening in the individual teststrips. In preferred embodiments, the gap has a width of between 1.0 mmand about 1.6 mm. As noted above, however, the gap could be replaced byusing a unitary covering layer having a notch formed on its underside(FIG. 1B) or by having the chamber cover overlap the body cover or viceversa. (FIG. 1C).

With further reference to FIG. 13, assembly 260 is ready for furtherprocessing as indicated by dashed lines 262 and 264. In FIG. 14 there isshown the kiss-cut strip 276 having a plurality of individual teststrips, e.g., 278 a, 278 b, and 278 c, detachably connected together. Itcan be observed that kiss-cut strip 276 has been trimmed or cut at itsupper end along lines 262 in FIG. 13 to have a profile and/orconfiguration suitable to facilitate capturing a very small fluid samplein each of the series of capillary channels 222. In the illustratedembodiment, kiss-cut strip 276 has a flat working end 280 exposing theend of the sets of Y-cut capillary channels 222. The resultingconfiguration of second edge 282 can be provided to facilitate insertionof a single strip into a meter (not shown). For example, the second edge282 can have a registration mark and/or tabs, cut slots, or otherconfigurations designed to allow insertion of a single strip into themeter in only one direction. For example, with reference to FIG. 13, theedges 177 of contact pads 288 are spaced by a constant pitch, “P” asshown, and edges 177 can therefore be used as registration marks. As inother processing steps, the indexing or registration marks 176 and 177on either the first edge and/or the second edge can be used toaccurately “kiss cut” and trim the individual test strips from thelaminated structure 260.

FIG. 15 is a perspective view of one embodiment of a punched cut teststrip 290 formed by cutting through the dashed lines 264 shown in FIGS.13 and 14. Strip 290 illustrated in FIG. 15 has been substantiallydescribed above as test strip 10. Strip 290 is provided as an individualtest strip separate from any other test strip.

Flared Portion of Sample Receiving Chamber

As described above and in more detail in this section, embodimentsincorporating the invention recited in the appended claims include asample receiving chamber having a flared portion that terminates in asample receiving opening. As one should appreciate, the capillary actionthat draws sample fluid into the capillary-sized sample receivingchamber is the result of adhesion of the fluid to the walls of thecapillary channel as well as the surface tension of the fluid to besampled. Adhesion of the fluid to the walls of the capillary channelresults in a force acting on the fluid at its edges and results in ameniscus. The surface tension, or cohesion of the liquid molecules ofthe fluid, acts to hold the surface intact, so instead of the edgesmoving inward in the capillary, the entire liquid sample is pulled intothe capillary. Capillary action occurs when the adhesion to the walls ofthe capillary channel is stronger than the cohesive forces between theliquid molecules in the bodily fluid sampled.

In a uniform capillary tube, the height to which capillary action willbe able to lift liquid depends on the surface tension of the liquid andthe weight of the liquid. By reducing the size of the capillary, theratio of adhesion of the fluid to the surface of the capillary to theweight or mass of liquid to be drawn into the capillary is increased,thereby increasing the net force which pulls the fluid into thecapillary. Consequently, capillaries that are smaller in size are ableto draw liquid more quickly and to a greater extent as compared tolarger capillaries. Thus, capillary channels are typically very smalland are continually being designed smaller to lessen the amount ofsample needed for testing.

However, the smaller the capillary entrance width, the more difficult itbecomes to accurately apply (or “target”) a 0.05 μl or 1 μl samplevolume to the capillary of the test strip. In the embodiments describedbelow, the capillary channels are flared or “Y-shaped”; i.e., theynarrow in a direction inwardly of the sample receiving opening.Advantageously, because the capillary channel narrows beyond the openingor entrance, the increased forces provided by the narrow and elongatedpart of the channel will draw fluid from the flared portion, which canalso be referred to and thought of as a “pre-chamber.” Furthermore, thispre-chamber acts as a virtual “reservoir,” which minimizes underdosingand minimizes the amount of sample that must be supplied to the strip.

A phenomena in which a sample is applied to the opening of a test stripbut hesitates to be drawn into the capillary is known as dosehesitation. As should be appreciated, this dose hesitation increases thetime required in order to collect an adequate sample. Extending thehydrophilic reagent layer to the dosing end of the test strip andcoating the underside of the chamber cover with a hydrophilic reagentreduces dose hesitation and promotes good wicking of the fluid sampleinto the capillary. Reducing dose hesitation in turn reduces fluidcollection times and makes fluid collection easier.

The capillary channels in the embodiments illustrated in FIGS. 16-19 areflared or “Y-shaped.” The flared portion provides a wider target areafor depositing a fluid sample onto the test strip. Further, the dosingend 36 of a test strip or biosensor is tapered to form a trapezoidalshaped profile in which a dosing edge 39 (FIG. 1) of the test strip isnarrower than the width of the remainder of the test strip. The taperedend reduces the length of the edge that is wasted, i.e., the portion ofthe edge that sample fluid can contact but not enter thesample-receiving chamber. Additionally, the Y-shaped opening increasesthe target area for the fluid sample. The combination of this taperedend and flared portion produces a synergistic effect, in that itprovides a test strip that will draw sample fluid into the samplereceiving chamber no matter where along the dosing edge 39 the samplemakes contact. This greatly reduces the chance of user error in dosingthe strip. Further, it is not necessary to cover the entire opening withsample fluid. This feature greatly facilitates the use of small liquidvolumes.

Turning now to FIG. 16A, a test strip or biosensor 300 is shown, whichis substantially identical to biosensor 10 illustrated hereinabove. FIG.16B illustrates a portion of a precursor or web 302, which correspondsto the structure shown in FIG. 12 after the chamber cover 240 and bodycover 234 have been laminated. As shown in FIG. 16B, chamber cover 304is clear and is spaced from body cover 306. Chamber cover 304 overliesthe spacer layer, which has been formed with a series of voids 308. Thevoids include an elongated portion 310 and a bulbous portion 312. Thebulbous portion can take a variety of shapes and comprises a shape whichultimately determines the shape of the flared opening, as described inmore detail below. The spacing or spacer layer from which voids 308 areformed in FIG. 16B is similar to that shown in FIGS. 10-12, describedabove. That is, the void extends all of the way to the edge of thespacing layer and therefore defines a discontinuous periphery. In otherwords, the void extends to the edge of the spacing layer.

By contrast, as shown in FIG. 16C, an alternate set of voids 314 havingcontinuous peripheries is formed in the spacing layer of precursor orweb 315. Voids 314 comprise a light bulb shape, having bulbous portions316 and elongated portions 318. In either case, the dosing edge of thetest strip is formed by cutting through the precursor along dashed line320 (FIG. 16B) or dashed line 322 (FIG. 16C), thereby forming thestructure 260 shown in FIG. 13. As the cut is made, preferably byshearing across the web in the direction indicated in FIG. 13, the cutextends or spans across the bulbous portion of the void. Placement ofthe cut (lines 320 and 322) should be precise with respect to thelongitudinal or lengthwise direction of the test strips ultimatelyformed from the web, such that the volume of the capillaries of thecompleted biosensors is consistent and within a tight tolerance. Ofcourse, misplacement of the dosing edge affects capillary volume morewhen the capillary or sample receiving chamber has a flared portion thanwhen it has parallel walls. Cutting along lines 320 and 322 not onlyforms the dosing end of the biosensors, but also forms the samplereceiving openings of the test strips, which openings are aligned withthe dosing edges. It should also be appreciated that while the dosingedge is shown in the illustrated embodiments as formed on the end of thetest strips, it could also be disposed on a side, for example.

After the dosing edge of the web is formed, the web may be cut alonglines 324 to form individual biosensors or test strips 300. As shown inFIG. 16A, biosensor 300 has a capillary or sample receiving chamber thatincludes a flared portion 328 that terminates in a sample receivingopening 330. An elongated portion 332 extends inwardly from the flaredportion. As shown in FIGS. 16A-16C, the elongated portion is formed ofsubstantially parallel walls that are defined by the void in the spacinglayer, while the walls of the flared portion angle outwardly orlaterally as the flared portion extends away from the elongated portion.The capillary or sample receiving chamber 326 communicates with ventopening 334 that is formed as a gap between chamber cover 304 and bodycover 306, as also described elsewhere.

It should also be appreciated that, in preferred embodiments, the cutalong lines 320 and 322 of FIGS. 16B-C also cuts through the reagentlayer (see FIGS. 10-14), such that the reagent layer extends to and iscoextensive with the dosing edge of the biosensors, as can beappreciated with respect to FIG. 2 discussed elsewhere herein. Since thereagent is hydrophilic, this advantageously promotes wicking of thesample into the biosensor and thus further discourages dose hesitation.Additionally, the cut forming the dosing edge also advantageouslyremoves the most uneven portion of the reagent layer, thereby leavingbehind an extremely flat and uniform reagent layer in the samplereceiving chamber.

By way of non-limiting example only, the flared portion 328 can have alength of about 0.80+/−0.2 mm; a width at the sample receiving openingof about 2.9 mm; and the flared walls form an angle of about 110°. Othersuitable dimensions for the sample receiving chamber are listedhereinabove. One of skill in the art should readily appreciate that thedimensions just noted are given merely as an example, and the dimensionsof a biosensor falling within the limits of the appended claims couldvary widely from those just given.

Indeed, many other shapes for the flared portion are possible. Forexample, turning now to FIGS. 17A-17C, biosensor 350 is similar tobiosensor 300 shown in FIG. 16A, except that the flared portion 352 ofsample receiving chamber 354 has curved walls rather than straight ones.In FIG. 17B, the voids 356 are formed as chalice shaped, whereas thevoids 358 in FIG. 17C are shaped like keyholes. In either event, thecuts along dashed lines 362 form the dosing edge of the strips that willin turn be formed by cutting along lines 364.

Similarly, turning to FIGS. 18A-18C, biosensor 400 is similar tobiosensor 300 shown in FIG. 16A, except that the flared portion 402 ofsample receiving chamber 404 has a T-shape. In FIG. 18B, the voids 406are formed as T-shaped, and the voids 408 in FIG. 18C are also T-shaped,having thinner cross-members of the “T.” In either event, the cuts alongdashed lines 410 (FIG. 18B) and 412 (FIG. 18C) form the dosing edge ofthe strips that will in turn be formed by cutting along lines 364.

Referring to FIG. 1C, an optional notch 41 can be formed at the dosingend of test strip 10. Notch 41 may help reduce dose hesitation andprovides a tactile sensation to the user that a finger or otheralternate site is located correctly with respect to biosensor 10 whendepositing a blood sample. The notch is disposed centrally with respectto the fluid receiving opening and is formed by cutting the same sizeand shape cut-out portions 43 and 45 in the aligned edges of coveringlayer 16 and base substrate 12, respectively. Cut-out portions 43 and 45align with one another as shown.

FIGS. 19A-19C illustrate various alternate embodiments of test strips orbiosensors having flared portions and/or notches that can be formed withthe present invention. In FIG. 19A, an end of a biosensor 500 is shownin which the dosing end 502 comprises a flat or straight edge. Samplereceiving chamber 504 includes a Y-shaped flared portion 506 leadinginwardly on the strip to an elongated portion 508. An optional V-shapednotch 510 can be cut through the covering layer and base substrate. InFIG. 19B, an end of a biosensor 512 is shown in which the dosing end 514comprises a curved profile. Sample receiving chamber 516 includes acurved shaped flared portion 518 leading inwardly on the strip to anelongated portion 520. An optional curved notch 522 can be cut throughthe covering layer and base substrate. Finally, in FIG. 19C, an end of abiosensor 524 is shown in which the dosing end 526 comprises a curvedconcave profile. Sample receiving chamber 528 includes a Y-shaped flaredportion 530 leading inwardly on the strip to an elongated portion 532.An optional V-shaped notch 534 can be cut through the covering layer andbase substrate.

EXAMPLE

By way of specific example, a test strip is formed based on thedescribed method and using materials as follows. The bottom substrate issurface coated with a 50 nm layer of gold, and is slit to widths of43-45 mm. Laser ablation (308 nm) is performed using a field size ofapproximately 40 mm×10 mm. The spacing layer assembly includes a spacinglayer film of white Melinex™ 339, and a thickness of 0.1016 or 0.127 mm(4 or 5 mil). The bottom and top adhesives are an Adhesive ResearchArcare 90132 adhesive at 0.0254 or 0.0127 mm (1 or ½ mil), sandwichedbetween release liners having a thickness of 0.0508 mm (2 mil). Thecapillary channels are formed with a width of 1.500 mm, +/−0.050 mm, anda pitch (spacing) of 9 mm, +/−0.150 mm.

The body cover 18 comprises a strip of Melinex 454, 453 or 339 material,0.127 mm (5 mil) thick. The chamber cover 20 comprises a polyester orpolyethylene naphthate material formed, for example, from Melinex 454 or453, 0.1016 mm (4 mil) thick. As indicated, the chamber cover may bepreferably treated or coated to have a hydrophilic underside adjacent tothe capillary channel to promote wicking of the blood specimen into thechannel. In a preferred embodiment, a Melinex 453 foil (4 mil) forchamber cover 20 is coated on its underside with a hydrophilic material21, ARCare 90037 from Adhesives Research Inc. Preferably the chambercover material is initially formed as a wider material, and is slit tothe desired width after preparation.

Test Strip Examples

The following materials will be used in the strip: Base substrate layer12 Melinex 329-9 mil or 329 - 10 mil Conductive Layer 26 Sputteredgold - 50 nm Lower Adhesive Layer 49 AR ARCare 90132 PSA - 1 to 0.5 milSpacing layer 14 Melinex 329 or 339 - 4 to 5 mil Adhesive Layer 46 ARARCare 90132 PSA - 1 to 0.5 mil Body Cover 18 Melinex 339 or 329 or454 - 5 mil Chamber Cover 20 Melinex 339 or 329 or 454 - 4 milHydrophilic foil 21 ARCare 90037Storage of Strips

Strips may be packaged in a variety of ways. For example, strips may bepackaged into flip-top plastic vials (e.g., 10, 25 or 50 count). Allcontainers include desiccant materials necessary to ensure acceptableshelf-life. Test strips preferably display a minimum shelf life of 18months when stored between 4°-32° C. in tightly closed containers asprovided.

While preferred embodiments incorporating the principles of the presentinvention have been disclosed hereinabove, the present invention is notlimited to the disclosed embodiments. Instead, this application isintended to cover such departures from the present disclosure as comewithin known or customary practice in the art to which this inventionpertains and which fall within the limits of the appended claims.

1. A test strip for analyzing a fluid, comprising: a test strip bodydefining a sample receiving chamber having a flared portion terminatingin a fluid receiving opening and an elongated portion extending inwardlyfrom the flared portion; the test strip body defining a vent incommunication with the sample receiving chamber, whereby air can escapefrom the vent as fluid is drawn into the sample receiving chamber; and atesting area positioned in the sample receiving chamber for analyzingthe fluid.
 2. The test strip of claim 1, wherein the flared portion isdefined by a pair of opening walls that narrow in a direction toward theelongated portion.
 3. The test strip of claim 1, wherein the flaredportion is defined by a pair of curved opening walls.
 4. The test stripof claim 1, wherein: the elongated portion is defined by a pair ofsubstantially parallel opposing sidewalls and an end wall connecting thesidewalls; and the flared portion is defined by a pair of walls thatnarrow in a direction toward the elongated portion.
 5. The test strip ofclaim 1, the test strip body further comprising: a base substrate; acovering layer overlying the base substrate; and a spacing layersandwiched between the base substrate and the covering layer, thespacing layer defining a void that defines the perimeter of the samplereceiving chamber between the base substrate and the covering layer. 6.The test strip of claim 5, wherein the vent is formed in the coveringlayer.
 7. The test strip of claim 6, wherein the covering layercomprises two pieces separated by a gap which overlies the samplereceiving chamber and defines the vent.
 8. The test strip of claim 5,wherein the covering layer, the spacing layer and the base substrate areall substantially flat.
 9. The test strip of claim 5, wherein thecovering layer and the base substrate include substantially alignededges that comprise a fluid receiving end of the test strip at which thefluid receiving opening is disposed.
 10. The test strip of claim 9,wherein the aligned edges are substantially straight.
 11. The test stripof claim 9, wherein the aligned edges comprise a convex profile.
 12. Thetest strip of claim 9, wherein the aligned edges are curved.
 13. Thetest strip of claim 9, wherein the aligned edges comprise a notch whichfurther defines the opening.
 14. The test strip of claim 1, wherein thesample receiving chamber has a volume less than or equal to 1.0 μl. 15.The test strip of claim 1, wherein the sample receiving chamber has avolume of less than or equal to 0.5 μl.
 16. The test strip of claim 1,wherein the sample receiving chamber includes at least one electrode anda reagent layer covering the at least one electrode.
 17. The test stripof claim 16, wherein the reagent layer extends to the sample receivingopening.
 18. The test strip of claim 1, wherein the fluid receivingopening is disposed at a tapered fluid receiving end of the test strip.19. The test strip of claim 18, wherein the fluid receiving endcomprises two sides that angle toward one another and terminate in adosing edge that is narrower than a width of the test strip.
 20. Thetest strip of claim 19, wherein the dosing edge is substantiallystraight.
 21. A test strip, comprising: a base substrate; a coveringlayer overlying the base substrate and comprising a vent; a spacinglayer disposed between the covering layer and the base substrate, thespacing layer having a void that defines a sample receiving chamberdisposed between the base substrate and the covering layer; and thesample receiving chamber defining a flared portion that terminates in afluid receiving opening.
 22. The test strip of claim 21, wherein thecovering layer, the base substrate and the spacing layer aresubstantially flat, and further wherein the sample receiving chambercomprises a substantially constant height and a width that varies at theflared portion.
 23. The test strip of claim 22, wherein the samplereceiving chamber includes an elongated portion having a substantiallyconstant width extending from the flared portion.
 24. The test strip ofclaim 21, wherein the sample receiving chamber includes an elongatedportion having a substantially constant width extending from the flaredportion.
 25. The test strip of claim 21, wherein the vent is positionedproximate a terminal end of the sample receiving chamber.
 26. The teststrip of claim 21, wherein the covering layer and the base substrateinclude substantially aligned edges that comprise a fluid receiving endor side of the test strip at which the fluid receiving opening islocated.
 27. The test strip of claim 26, wherein the aligned edges aresubstantially straight.
 28. The test strip of claim 26, wherein thealigned edges comprise a convex profile.
 29. The test strip of claim 26,wherein the aligned edges are curved.
 30. The test strip of claim 26,wherein the aligned edges comprise the fluid receiving end.
 31. The teststrip of claim 26, wherein the aligned edges comprise a notch whichfurther defines the opening.
 32. The test strip of claim 31, wherein thenotch is smaller than the flared portion.
 33. The test strip of claim31, wherein the notch and flared portion have substantially matchingshapes.
 34. The test strip of claim 31, wherein the notch is disposedcentrally with respect to the flared portion.
 35. The test strip ofclaim 21, further comprising at least one electrode and a reagent layerdisposed in the sample receiving chamber, the reagent layer covering theat least one electrode.
 36. The test strip of claim 35, wherein thereagent layer extends to the sample receiving opening.
 37. The teststrip of claim 21, wherein the fluid receiving opening is disposed at atapered fluid receiving end of the test strip.
 38. The test strip ofclaim 37, wherein the fluid receiving end comprises two sides that angletoward one another and terminate in a dosing edge that is narrower thana width of the test strip.
 39. The test strip of claim 38, wherein thedosing edge is substantially straight.
 40. A test strip, comprising: abase substrate having a reagent layer disposed thereon; a covering layeroverlying the base substrate and comprising a chamber cover and a bodycover having a slot therebetween; a sample receiving chamber disposedbetween the base substrate and the covering layer, the slot being incommunication with the sample receiving chamber and defining a ventopening in the covering layer that allows air to escape as fluid entersthe sample receiving chamber; and wherein the sample receiving chambercomprises a flared portion defining a sample receiving opening.
 41. Thetest strip of claim 40, wherein the slot comprises a gap which spacesthe body cover from the chamber cover.
 42. The test strip of claim 40,wherein the sample receiving chamber comprises an elongated portionextending inwardly from the flared portion.
 43. The test strip of claim42, wherein the flared portion is defined by a pair of opening wallsthat narrow in a direction toward the elongated portion.
 44. The teststrip of claim 42, wherein the flared portion is defined by a pair ofcurved opening walls.
 45. The test strip of claim 42, wherein: theelongated portion is defined by a pair of substantially parallelopposing sidewalls and an end wall connecting the sidewalls; and theflared portion is defined by a pair of walls that narrow in a directiontoward the elongated portion.
 46. The test strip of claim 40, whereinthe covering layer and the base substrate include substantially alignededges that comprise a fluid receiving end or side of the test strip atwhich the sample receiving opening is disposed.
 47. The test strip ofclaim 46, wherein the aligned edges are substantially straight.
 48. Thetest strip of claim 46, wherein the aligned edges comprise a convexprofile.
 49. The test strip of claim 46, wherein the aligned edges arecurved.
 50. The test strip of claim 46, wherein the aligned edgescomprise a notch which further defines the sample receiving opening. 51.The test strip of claim 46, wherein the aligned edges comprise the fluidreceiving end of the test strip.
 52. The test strip of claim 40, furthercomprising an electrode disposed in the sample receiving chamber, theelectrode being covered by the reagent layer.
 53. The test strip ofclaim 40, wherein the chamber cover overlies the sample-receivingchamber.
 54. The test strip of claim 40, further comprising a spacinglayer disposed between the covering layer and the base substrate, thespacing layer defining a void portion that further defines the heightand perimeter of the sample receiving chamber between the base substrateand the covering layer.
 55. The test strip of claim 54, wherein the voidportion defines the flared portion and further defines an elongatedportion extending inwardly from the flared portion.
 56. The test stripof claim 54, wherein the void terminates at a position substantiallyaligned with the slot.
 57. The test strip of claim 40, wherein thesample receiving opening is disposed at a tapered fluid receiving end ofthe test strip.
 58. The test strip of claim 57, wherein the fluidreceiving end comprises two sides that angle toward one another andterminate in a dosing edge that is narrower than a width of the teststrip.
 59. The test strip of claim 58, wherein the dosing edge issubstantially straight.
 60. A test strip, comprising: a base substratehaving a reagent layer disposed thereon; a covering layer overlying thebase substrate; a sample receiving chamber disposed between the basesubstrate and the covering layer, the sample receiving chambercomprising a flared portion terminating in a sample receiving openinglocated at an edge of the test strip; and wherein the reagent layer isdisposed in the flared portion and extends to the opening located at theedge of the test strip.
 61. The test strip of claim 60, furthercomprising a slot in communication with the sample receiving chamber,the slot defining a vent opening in the covering layer that allows airto escape as fluid enters the sample receiving chamber.
 62. The teststrip of claim 60, wherein the sample receiving chamber comprises anelongated portion extending inwardly from the flared portion.
 63. Thetest strip of claim 62, wherein the flared portion is defined by a pairof opening walls that narrow in a direction toward the elongatedportion.
 64. The test strip of claim 62, wherein the flared portion isdefined by a pair of curved opening walls.
 65. The test strip of claim62, wherein: the elongated portion is defined by a pair of substantiallyparallel opposing sidewalls and an end wall connecting the sidewalls;and the flared portion is defined by a pair of walls that narrow in adirection toward the elongated portion.
 66. The test strip of claim 60,wherein the covering layer and the base substrate include substantiallyaligned edges that comprise a fluid receiving end or side of the teststrip at which the fluid receiving opening is disposed.
 67. The teststrip of claim 66, wherein the aligned edges are substantially straight.68. The test strip of claim 66, wherein the aligned edges comprise aconvex profile.
 69. The test strip of claim 66, wherein the alignededges are curved.
 70. The test strip of claim 66, wherein the alignededges comprise a notch which further defines the sample receivingopening.
 71. The test strip of claim 66, wherein the substantiallyaligned edges comprise the fluid receiving end of the test strip. 72.The test strip of claim 60, further comprising an electrode disposed inthe sample receiving chamber, the electrode being covered by the reagentlayer.
 73. The test strip of claim 60, further comprising a spacinglayer disposed between the covering layer and the base substrate, thespacing layer defining a void portion that further defines the heightand perimeter of the sample receiving chamber between the base substrateand the covering layer.
 74. The test strip of claim 73, wherein theheight is substantially constant throughout the sample receivingchamber.
 75. The test strip of claim 60, wherein the sample receivingopening is disposed at a tapered fluid receiving end of the test strip.76. The test strip of claim 75, wherein: the fluid receiving endcomprises two sides that angle toward one another and terminate in theedge at which the sample receiving opening is located; and the edge isnarrower than a width of the test strip.
 77. The test strip of claim 76,wherein the edge is substantially straight.
 78. A method of making atest strip, comprising: (a) providing a base substrate, a spacing layerand a covering layer; (b) forming a void in the spacing layer, the voidhaving an elongated portion and a bulbous portion; (c) laminating thespacing layer to the base substrate and laminating the covering layer tothe spacing layer, thereby forming a test strip precursor; and (d)cutting through the precursor to make the test strip, the cut crossingthe bulbous portion of the void and forming a sample receiving edge ofthe test strip, wherein the void defines a sample receiving chamberhaving a flared portion terminating in a sample receiving opening at thesample receiving edge of the test strip.
 79. The method of claim 78,wherein the sample receiving edge comprises an end of the test strip.80. The method of claim 78, wherein the sample receiving edge comprisesa side of the test strip.
 81. The method of claim 78, wherein the voidformed in step (b) has a continuous periphery.
 82. The method of claim78, wherein the void formed in step (b) has a discontinuous periphery.83. The method of claim 78, wherein the flared portion is Y-shaped. 84.The method of claim 78, further comprising an elongated portion of thesample receiving chamber extending inwardly from the flared portion. 85.The method of claim 78, wherein the void extends through the spacinglayer, wherein the sample receiving chamber is bounded by the coveringlayer and the base substrate.
 86. The method of claim 78, wherein: thebase substrate comprises a web of base substrate material, the spacinglayer comprises a web of spacing layer material and the covering layercomprises a web of covering layer material; the void formed in step (b)comprises a plurality of voids spaced apart; and the method furthercomprises cutting the precursor into a plurality of test strips.
 87. Themethod of claim 78, further comprising printing a graphic onto the teststrip precursor.
 88. The method of claim 87, further comprisingperforming the printing the graphic onto the test strip precursor afterperforming step (d).
 89. The method of claim 78, wherein the samplereceiving edge is positioned at a dosing end of the test strip and thedosing end is tapered.
 90. The method of claim 89, wherein the dosingend comprises two sides that angle toward one another and terminate in adosing edge that is narrower than a width of the test strip.
 91. Themethod of claim 90, wherein the dosing edge is substantially straight.92. A test strip, comprising: a base substrate having a reagent layerdisposed thereon; a covering layer overlying the base substrate; asample receiving chamber disposed between the base substrate and thecovering layer, the sample receiving chamber comprising a flared portionterminating in a sample receiving opening at a dosing end of the teststrip; and wherein the dosing end is tapered.
 93. The test strip ofclaim 92, wherein the reagent layer is disposed in the flared portionand extends to a dosing edge of the dosing end.
 94. The test strip ofclaim 92, wherein the covering layer comprises a chamber cover overlyingthe sample receiving chamber.
 95. The test strip of claim 94, whereinthe chamber cover further comprises a hydrophilic coating disposedthereon, the hydrophilic coating defining a ceiling of the samplereceiving chamber.
 96. The test strip of claim 94, wherein a portion ofthe chamber cover facing the sample receiving chamber is hydrophilic.97. The test strip of claim 96, wherein the reagent layer extends to adosing edge of the dosing end.
 98. The test strip of claim 94, whereinthe chamber cover is hydrophilic.
 99. The test strip of claim 98,wherein the reagent layer extends to a dosing edge of the dosing end.100. The test strip of claim 94, wherein the chamber cover comprises asubstantially transparent material.
 101. The test strip of claim 92,wherein the dosing end comprises two sides that angle toward one anotherand terminate in a dosing edge that is narrower than a width of the teststrip.
 102. The test strip of claim 101, wherein the dosing edge issubstantially straight.
 103. The test strip of claim 92, wherein thedosing end comprises a curved shape.
 104. The test strip of claim 92,further comprising a spacing layer disposed between the base substrateand the covering layer, the spacing layer defining a void that defines asample receiving chamber between the base substrate and the coveringlayer.
 105. The test strip of claim 104, wherein the spacing layercomprises a first color and the reagent layer comprises a second colorthat contrasts with the first color.
 106. The test strip of claim 105,wherein the covering layer is substantially transparent over the samplereceiving chamber, whereby the contrasting colors of the reagent layerand the spacing layer are visible through the covering layer.
 107. Thetest strip of claim 106, wherein the first color and the second colorcontrast with blood.
 108. The test strip of claim 105, wherein thecovering layer comprises a chamber cover, the chamber cover issubstantially transparent and the contrasting colors of the reagentlayer and the spacing layer are visible through the chamber cover. 109.The test strip of claim 108, wherein the first color and the secondcolor contrast with blood.
 110. The test strip of claim 104, furthercomprising a graphic disposed on the spacing layer.
 111. The test stripof claim 110, wherein the graphic comprises a chevron.
 112. A teststrip, comprising: a base substrate having a reagent layer disposedthereon, the reagent layer comprising a first color; a covering layeroverlying the base substrate; a spacing layer disposed between the basesubstrate and the covering layer, the spacing layer defining a void thatdefines a sample receiving chamber between the base substrate and thecovering layer, the spacing layer comprising a second color thatcontrasts with the first color; and the sample receiving chambercomprising a flared portion terminating in a sample receiving opening ata dosing end of the test strip.
 113. The test strip of claim 112,wherein the covering layer comprises a chamber cover overlying thesample receiving chamber.
 114. The test strip of claim 113, wherein thechamber cover comprises a transparent or translucent material, wherebythe contrasting colors of the reagent layer and spacing layer arevisible through the chamber cover.
 115. The test strip of claim 114,wherein the contrasting colors of the reagent layer and the spacinglayer contrast with blood.
 116. The test strip of claim 113, wherein thechamber cover comprises a hydrophilic underside.
 117. The test strip ofclaim 112, further comprising a graphic disposed on the spacing layer.118. The test strip of claim 117, wherein the graphic comprises achevron.
 119. The test strip of claim 112, wherein the dosing end istapered.
 120. The test strip of claim 119, wherein the dosing endcomprises two sides that angle toward one another and terminate in adosing edge that is narrower than the width of the test strip.
 121. Thetest strip of claim 120, wherein the dosing edge is substantiallystraight.
 122. The test strip of claim 112, wherein the reagent layerextends to a dosing edge of the test strip.